Bioprinting of Stem Cells: Interplay of Bioprinting Process, Bioinks, and

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Bioprinting of stem cells: interplay of bioprinting process, bioinks and stem cell properties. Supeng Ding, Lu Feng, Jiayang Wu, Fei Zhu, Ze'en Tan, and Rui Yao ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00399 • Publication Date (Web): 25 Jul 2018 Downloaded from http://pubs.acs.org on July 27, 2018

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ACS Biomaterials Science & Engineering

Bioprinting of stem cells: interplay of bioprinting process, bioinks and stem cell properties Supeng Ding 1,2, Lu Feng1, Jiayang Wu 1,3, Fei Zhu 1, Ze’en Tan 1, Rui Yao 1*

1 Department of Mechanical Engineering, Biomanufacturing and Rapid Forming Technology Key Laboratory of Beijing, Tsinghua University, Beijing, People’s Republic of China 2 Department of Materials Science and Engineering, Tsinghua University, Beijing, People’s Republic of China 3 Department of Construction Management, Tsinghua University, Beijing, People’s Republic of China

Corresponding Author*:

Rui Yao: [email protected]

Abstract Combining the advantages of 3D bioprinting technology and biological characteristics of stem cells, bioprinting of stem cells is recognized as a novel technology with broad applications in biological study, drug testing, tissue engineering and regenerative medicine, etc. However, the biological performance and functional reconstruction of stem cells are greatly influenced by both the bioprinting process and post-bioprinting culture condition, which are critical factors 1

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to consider for further applications. Here we review the recent development of stem cell bioprinting technology and conclude major factors regulating stem cell viability, proliferation, differentiation and function from the aspects of the choice of bioprinting techniques, the modulation of bioprinting parameters and the regulation of stem cell niche in the whole lifespan of bioprinting practice. We aim to provide a comprehensive consideration and guidance of bioprinting of stem cells for optimization of this promising technology in biological and medical application.

Keywords: 3D Printing, Biofabrication, Multipotent Stem Cells, Pluripotent Stem Cells, Stem Cell Niche Regulation, Long-term Culture

1. Introduction It has become a consensus that research on stem cells is of great significance in medicine, physiology and biology study since they demonstrate unique abilities of proliferation and differentiation compared with mature primary cells. Based on these features, stem cell technology has been widely applied in the treatment of disease 1,2, drug development 2,3, transgenic production 4, the pathological study 5, and has revealed potentials in organ transplantation and regenerative medicine 2, etc. Nevertheless, being sensitive to the microenvironment, stem cell properties can be influenced by many physical and chemical factors, causing low viability, limited proliferation and undesirable differentiation in many cases 1,4,6-8. This has become a primary obstacle in the development and application of stem 2


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cell technology. Therefore, there is a pressing need for technologies offering specific design of stem cell niche with the mild procedure to satisfy both the essential biological requirements of stem cells and directed differentiation to desired lineages.

Bioprinting technology is commonly known as the process of creating cell and biomaterial patterns in a confined space using 3D printing technique, where cell function and viability are preserved within the printed construct 9,10. To realize transition from printable fluid to long-term stable structure, cells are encapsulated within bioinks, which refer to biomaterials with the ability to be deposited by certain bioprinting techniques and providing the microenvironment for cell adhesion, proliferation and differentiation after bioprinting 11. In 2015, “bioprinting” became an official term in the Oxford Dictionary. It was defined as the use of 3D printing technology with materials that incorporate viable living cells, e.g. to produce tissue for reconstructive surgery. This technology can serve as one of the solutions for the construction of geometrically, physically and chemically controlled stem cell microenvironment. Compared with traditional methods for constructing 3D cell-laden structures, such as hanging-drop 12, multi-well plate 13, and stamping 14, bioprinting technology has an irreplaceable advantage of building delicate, complex and well-designed microstructure.

Bioprinting process is applicable for different purposes because of adjustable bioprinting principle and parameters, including printing speed, pressure, resolution, etc 15. Various bioinks can be used for inducing stem cell fate by offering physical and chemical cues 16,17, while 3



additional factors can be supplemented in the bioinks or culture media for further induction or maturation 18,19. As a result, stem cell bioprinting has become the rational choice by many researchers for either enhancing the understanding of stem cells or enlarging the range of application of both stem cell and bioprinting technology in tissue engineering, regenerative medicine, drug discovery, etc. According to the Pubmed database, 350 articles are available by searching with the keywords ‘(bioprinting OR printing) AND stem AND cell’ in recent 5 years, with a trend of continuous growth in recent years, indicating the booming of this field.

However, both bioprinting process and subsequent culture condition might lead to unwanted effects on stem cells properties. Several factors such as mechanical stress 20, working temperature 21, radiation 22 and bioink properties 23-26 are associated with cell injury and undesired differentiation when administrated improperly. Therefore, it is necessary to emphasize understanding of the underlying interactions among bioprinting process, bioinks and stem cells in the whole lifespan of stem cell bioprinting practice. Bioprinting process should satisfy the requirement of fabrication as well as stem cells at the same time. Bioinks should demonstrate printabilty in order to create delicate and stable structure, and also biocompatibility to induce stem cell differentiation and support their functions. Focusing on these interactions, we provide guidance and perspective about how to choose or even develop suitable bioprinting techniques, parameters and bioinks depending on the stem cell types and specific application.

Bioprinting factors related to stem cell behaviors are elucidated in three stages, namely: 4


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before bioprinting, during bioprinting and after bioprinting. Before bioprinting, suitable bioprinting techniques should be chosen based on types of stem cells, properties of the desired structure, and anticipated cost. During bioprinting, printing parameters ought to be optimized to mitigate harm to stem cells. After bioprinting, microenvironment created by bioinks and culture media can further regulate long-term cell proliferation and differentiation (Figure 1). Since bioinks play the leading role in establishment of delicate structure and creation of biocompatible microenvironment for stem cell culture, they are discussed specifically. We overview recent achievements in the whole lifespan of stem cell bioprinting and discuss potential challenges and improvements for future research.

Figure 1. Schematic of the whole lifespan of stem cell bioprinting practice. Reproduced with permission from ref 27, 28. Copyright 2015 and 2016 Biofabrication.

2. Choosing suitable bioprinting techniques In order to satisfy demands of various research, different bioprinting techniques have been 5



developed from perspectives of manufacturing abilities (printing resolution, speed, throughput, etc.) 29 and cell requirements (cell viability, proliferation, differentiation, etc.) 14. There are two ways of classifying bioprinting technology, which are not mutually exclusive. One way is based on the cell format (dispersed individual cells or cell aggregates) and cell number resulted from each printing motion (only one cell or more than one cell), bioprinting techniques can be mainly divided into multi-cell, single-cell and cell aggregate bioprinting. In multi-cell bioprinting, large quantities of dispersed individual cells are dispersed in bioinks and more than one cell is delivered within a droplet or a filament. In single-cell bioprinting, merely one cell is printed at each printing motion. In cell aggregate bioprinting, pre-formed cell aggregates instead of dispersed individual cells are embedded in the bioinks and then bioprinted.

Another method to classify bioprinting techniques is based on enabling technology, which are commonly seen in current reports, namely inkjet bioprinting, microextrusion-based bioprinting, laser-assisted bioprinting and stereolithography-based bioprinting. We choose to introduce the four enabling techniques under multi-cell bioprinting category, since compared with single-cell bioprinting and cell aggregate bioprinting, it is the most mature and developed field (Figure 2).

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Figure 2. Illustrations and examples of different bioprinting techniques. (A) Multi-cell bioprinting, which can be further divided into inkjet bioprinting (image from MicroFab Technologies, Inc.), microextrusion-based bioprinting (image from Organovo Holdings, Inc.), laser-assisted bioprinting (image from Organovo Holdings, Inc.) and stereolithography-based bioprinting (image from Formlabs, Inc.). (B) Cell aggregate bioprinting. Reproduced with permission from ref 30. Copyright 2009 7



Biomaterials. (C) Single-cell bioprinting. Reproduced with permission from ref 31. Copyright 2012 Biofabrication.

2.1. Multi-cell bioprinting 2.1.1. Inkjet bioprinting Inkjet bioprinting of cells was first reported by Roth E. A. et al. in 2004 to achieve high-throughput patterning of smooth muscle cells 32. Also known as drop-on-demand bioprinting, it is defined as dispensing through a small orifice and precise positioning of small volumes (1–100 picolitres) of bioink and/or cells on a substrate to fabricate 3D cell patterns or 3D cell-laden constructs 33. Thermal 34, piezoelectric 35, valve-based 36 and electric field-based inkjet bioprinting 37,38 are the most commonly adopted approaches. Thermal inkjet bioprinters function by heating the printing head to produce pulses of pressure that forces droplets out of the nozzle. In piezoelectric inkjet bioprinting, a mechanical pulse is applied to the fluid in the nozzle by a piezoelectric actuator causing a shock wave that forces the bioink throughout the nozzle. In electric field-based inkjet bioprinting, bioinks are forced to leave the nozzle by static electric force and then form a droplet in the crosslinking medium. Inkjet bioprinting demonstrates a series of advantages including low cost, high resolution, high printing speed and high post-bioprinting viability and has been employed on stem cells in recent years. Multipotent stem cells including mesenchymal stem cells (MSC) 39,40, muscle-derived stem cells 41, and neural stem cells (NSC) 42 have been bioprinted for regenerative medicine purposes. In addition, co-printing of amniotic fluid-derived stem cells and canine smooth muscle cells was reported by Xu T. et al., demonstrating the feasibility of establishing 8


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heterogeneous tissue structure from stem cells by inkjet bioprinting technology 43. As for pluripotent stem cells, Faulkner-Jones A. et al. bioprinted embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) to study stem cell differentiation in 3D microenvironment 21.

Physical and chemical factors involved in bioprinting process, including mechanical force, bioink viscosity and temperature should be paid attention to in the practice of inkjet bioprinting of stem cells. Stem cell injury and even death may result from both the shear stress by extrusion through small orifices of the print head 20 and/or the normal stress by reaching the substrate. Besides, bioinks used in inkjet bioprinting are confined to low viscosity to avoid frequent clogging. However, low viscosity bioink usually ends up with immediate droplet spreading on the substrate, impeding the building of designed 3D constructs with sound shape fidelity and mechanical stability 22. What’s more, the high temperature in thermal inkjet bioprinting can be a potential risk, although the duration of localized heating is short (~2µs) and little harm to cells has been reported 44. Xu T. et al. reported that thermal inkjet bioprinting could have a benign effect on simultaneous gene transfection by co-printing of porcine aortic endothelial cells (pAEC) and plasmids encoding green fluorescent protein 45. Further research on the influence of high temperature on cell biology and function in thermal inkjet bioprinting is still required.

2.1.2. Microextrusion-based bioprinting Microextrusion-based bioprinting, also known as bioplotting technology, was first reported by 9



Fedorovich N. E. et al. in 2007 that spatially organized constructs could be fabricated by microextrusion-based bioprinting of bone marrow stromal cell-laden alginate 46. In this technique, continuous filaments rather than liquid droplets of bioinks are extruded throughout a micro-nozzle to form 3D structures 15. One of the outstanding features is that the viscosity of bioinks used can range from 30 mPa·s to more than 6 × 107 mPa·s, providing much broader bioink options to meet requirements of different types of stem cells and provide desired mechanical strength 47. Besides, shear-thinning bioinks, which exhibit the non-Newtonian behavior of fluids whose viscosity decreases under shear strain, have been explored to further improve the printing resolution and shape fidelity of the 3D constructs 48. Other advantages includes high cell density 22 and high printing throughput, which enables fabrication of organized constructs with clinically relevant sizes 49,50. bioprinting is also recommended as an inexpensive bioprinting approach for industrial application in future, where Reid J. A. et al. invented a 3D printer with a cost less than $200 for iPSC bioprinting 51

.

In recent years, further optimization has been developed in this technique. Co-axial extrusion of multi-components with a microfluidic bioprinting head has been realized for reaction-based bioink crosslinking. Since only the surface of the filament is crosslinked and the inner bioinks remain liquid during bioprinting process, a core-shell structure of filament is generated, where the surface provides a good formability and the inner part with low viscosity ensures high cell viability 52,53,54. Multi-nozzle bioprinters can improve printing throughput and support deposition of multi-bioinks encapsulated with various cells, holding the potential of 10


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regenerating complex tissues 55,56. Moreover, Highley C. B. et al. explored a method to create constructs with complex structure by bioprinting a second bioink inside a support self-healing hydrogel 57. Based on its shear-thinning property, the self-healing hydrogel could deform when a syringe needle was inserted, but recovered rapidly to retain the printed structure. As the most common and affordable bioprinting techniques, bioprinting has been widely used in printing multipotent stem cells including bone marrow-derived mesenchymal stem cells (BMSC) 58, adipose-derived stem cells (ASC) 59,60, neural stem cells 61,62, and glioma stem cells 63. In recent years, more and more research has been focused on pluripotent stem cells. For example, Ouyang L. et al. utilized bioprinting technique to fabricate ESC-laden hydrogel structure with good printability and high cell viability, where printing temperature, holding time and bioink concentration can be controlled precisely 27. Moreover, it was reported by Gu Q. et al. that bioprinting of iPSCs could facilitate cell proliferation and successive differentiation 64.

Although widely used, several limitations need to be aware of in bioprinting research. Firstly, high bioink viscosity may reduce cell survival rate, mainly attributed to the shear stress inflicted on cells in viscous fluids 27,65. Secondly, extrusion pressure should be gentle enough to avoid increased cell death, which restricts further improvement of bioprinting speed and throughput. Thirdly, a decrease of nozzle size to achieve higher printing resolution can affect cell survival as well 66. Therefore, bioprinting process and parameters need to be carefully adjusted to achieve both high cell viability and sound printability.

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2.1.3. Laser-assisted bioprinting Laser-assisted bioprinting was first proposed by Ringeisen B. R. et al. in 2004, where pluripotent murine embryonal carcinoma cells embedded in matrigel were transferred to the substrate in an organized pattern with little cell damage 67. Based on the principles of laser-induced forward transfer (LIFT), laser pulses are focused on an energy-absorbing layer to provide energy for generating bubbles in another layer of cell-laden bioinks, which subsequently propel bioinks from the donor film to the receiving substrate 15,68,69. As a nozzle-free method, problems including bioink clogging and cell death resulted from shear stress and can be avoided, leading to higher cell viability. Besides, higher printing resolution can be achieved in this techniques, which is dependent on bioink viscosity, laser energy and scanning speed 70. Ali M. et al. reported laser-assisted bioprinting of MSCs with post-printing cell viability close to 100% 71. Sorkio A. et al. reported stroma-mimicking structures could be generated by laser-assisted bioprinting of ADSCs 72. Dias A. D. et al. investigated laser-assisted bioprinting of ESC, which served as an approach for the formation of the embryoid body 73.

Nevertheless, laser-assisted bioprinting is relatively time-consuming, rendering limitations in the high-throughput study since overlong printing time can reduce cell viability 16. Moreover, the cost of establishing laser-assisted bioprinting system is much higher than the above two techniques. Therefore, this technique is suggested to be applied in the fabrication of delicate structure of small size.

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2.1.4. Stereolithography-based bioprinting Stereolithography-based bioprinting was first used for establishment of human dermal fibroblasts-laden poly(ethylene glycol) (PEG) structure by Arcaute K. et al. in 2006 74. Liquid biocompatible resins are solidified by photopolymerization under irradiation in a designed pattern to fabricate layer-by-layer construct 75. As another nozzle-free technique, mechanic shear stress can be avoided. High bioprinting resolution is a noteworthy advantage where commercial systems usually create patterns with the resolution around 100µm~200µm. The resolution can even reach 50µm, in a case of bioprinting 3T3 fibroblast cells encapsulated in the mixture of polyethylene glycol diacrylate (PEGDA) and gelatin methacrylate (GelMA) hydrogel 76. Since crosslinking is realized by covalent binding, another advantage of stereolithography-based bioprinting is the long-term stability. As a consequence, constructs with fine pore structure can be fabricated, which is significant in the maintenance of mechanical properties and mass transfer for long-term cell culture 75. In stem cell research, stereolithography-based bioprinting has been employed in bioprinting multipotent stem cells such as BMSCs 77-80, ASCs 81, and NSCs 82, for enhanced differentiation or tissue repair. For pluripotent stem cells, Bajaj P. et al. reported that ESC encapsulated in PEGDA could be patterned by stereolithography-based bioprinting for 3D culture 83.

Bioinks utilized in Stereolithography-based bioprinting technology need to be biocompatible, biodegradable and photocurable, resulting in relatively limited choice. Commonly used bioinks includes PEGDA, poly(propylene fumarate) (PPF), PTMC, poly(e-caprolactone) (PCL), GelMA, and poly(D,L-lactide) (PDLLA). Combination of photocurable materials and 13



natural ECM matrix is also a common option to improve cell performance and/or induce desired differentiation and function 75,76,84. But it is reported that the long-term cell viability in stereolithography-based bioprinting is lower than the techniques above, since these bioinks have less biocompatibility and photoinitiators may decrease cell survival 85. Besides, the energy of UV light may result in cell death or induce carcinogenesis to some extent, which can be more serious in bioprinting of stem cells 76. Thus, the development of visible light crosslinking bioinks is a clear trend to broaden the perspective of this promising technology.

2.2. Cell aggregate bioprinting Unlike multi-cell bioprinting where single cells are individually distributed in the bioink, cell aggregate bioprinting utilizes the properties of cell aggregates to mimic the structure and function of tissues in vivo such as organoids 86. Cells in aggregates experience a divergent microenvironment comparing with single cells, since cell-cell contacts increase and cell-substrate contacts decrease. Coburn L. et al. modeled the cellular force distribution inside an epithelial cell aggregate and demonstrated that contact inhibition of locomotion can modulate the mechanical response of cells 87. In addition, immunomodulatory paracrine factor secretion increased in MSC aggregates due to the enhancement of intercellular adhesion and cell contact-dependent signaling 88. Komatsu M. et al. demonstrated that aggregates can assist maturation of dopamine neuron precursors into neurons 89. Meanwhile, cell aggregate bioprinting has a much higher throughput, since the number of cells in an aggregate commonly ranges from 500 to 250,000 90. Therefore, this technique is of significance for tissue regeneration of larger scale. 14


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The process for cell aggregate bioprinting includes the formation of cell aggregates, bioprinting of aggregates, and aggregate fusion and maturation. With self-assembly and self-organization, generation of cell aggregates can be realized by several methods, such as hanging-drop culture, non-adhesive surface, scaffolds, and microfabrication 91, with different aggregate shapes like spheroids, cylinders 92 and sheets 49. These cell aggregates should have the adequate mechanical strength to avoid fracture during bioprinting and long-term culture, which is an essential problem when considering the type of cells for cell aggregate bioprinting.

Subsequently, cell aggregates are printed by specific approaches, which can be mainly divided into two types based on cell-substrate contact. The first one is similar to multi-cell bioprinting where cell aggregates are encapsulated in bioactive bioinks, which provide adhesion ligands. For example, Marchioli G. et al. bioprinted islets encapsulated in a mixture of gelatin and alginate for islet transplantation 93. In the second one, namely scaffold-free bioprinting, bioinert materials like alginate 94 or agarose 31 and cell aggregates are co-printed. Cell aggregates are deposited adjacently without the barrier of materials and bioinks merely function as upholders before the cell aggregates complete the fusion, forming a tissue stable enough to hold itself.

After bioprinting, a quick fusion of cell aggregates is important for building stable tissues. Since cell aggregates serve as building blocks in the structure, the stability of the printed 15



structure can be enhanced via aggregate fusion by improving inter-aggregate force 92. This process is suggested to be regulated by cellular interaction and cell migration, and in some case analyzed by phase field theory 95,96. Maturation of cell aggregates is another vital process. To mimic the tissue formation in vivo, additional factors can be considered to modulate microenvironment of cell aggregates, accelerating maturation to generate stable and functional tissues 97.

2.3. Single-cell bioprinting Single-cell bioprinting aims to distribute singular cell in a controlled way for the fabrication of delicate tissues and single cell studies, such as genetic characterization of single cells 98,99. It is especially significant for stem cell research since the cells and microenvironment components can be accurately arranged.

Such an idea was first proposed in 2005 just after the invention of laser-assisted bioprinting, as Barron J. A. et al. reported laser-assisted bioprinting of human osteosarcoma cells. They utilized bioinks with different cell concentration to control the cell numbers in one droplet 100. However, although the size of droplets was homogeneous and small enough, one cell in each droplet was hardly realized and the cell number in one droplet basically obeyed Poisson distribution whatever the cell concentration was 100. Therefore, it was until 2012 that the real single-cell bioprinting had been invented, where the high-speed camera was used to check the cell status in the ejection area of the nozzle tip. When there was no cell or over one cell, the droplet was discarded 32. In 2014, a block-cell-printing technique was proposed by Zhang K. 16


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et al., where hook-shaped traps were positioned in designated patterns to trap singular breast cancer cell and work as printing nozzle 101. Although single-cell bioprinting of stem cells has not been reported as far as we know, there is a clear trend in the emerging of stem cell bioprinting technology assisted single cell study, due to the significance of stem cell biology.

In conclusion, to choose a suitable bioprinting technique, both fabrication process and cell requirements should be considered. Cell aggregate bioprinting is usually required in generation of tissues with a tight junction between cells and of great size. With the ability to build delicate structures, single-cell bioprinting is utilized in research on cellular-matrix interaction by depositing stem cells and microenvironment elements with precisely defined spatial location. On the other hand, multi-cell bioprinting is most widely used and most developed technology. Inkjet bioprinting and microextrusion-based bioprinting are cost efficient approach suitable for relatively big and rough structures while preserving high cell viability, while laser-assisted bioprinting can offer high resolution with little cell death. Stereolithography-based bioprinting can generate even more elaborate structures, but is less biocompatible. In addition, it is better to choose a technique with lower cost and gelation methods suitable for the specific lab equipment. In all, after a comprehensive consideration of the size, precision and complexity of desired structures and the type of cells, limitations can be determined from various aspects including printing speed, throughput, resolution, cell viability and density, based on which we can decide the suitable bioprinting technique. Table 1 can be used to help with choice of bioprinting technique.

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Table 1. Comparison of bioprinting techniques Multi-cell bioprinting Cell aggregate bioprinting

Single-cell bioprinting

High

Medium

High

Medium

High

High

Low

Techniques Inkjet bioprinting

Microextrusion-based bioprinting

Laser-assisted bioprinting

Stereolithography-based bioprinting

Printing speed

High

Low

Medium

Printing throughput

Medium

High

Resolution

Medium

Low

High

High

Low

High

Cell viability

Medium

Medium

High

Low

High

Medium

Cell density

Low

High

Medium

Medium

High

Low

Cost

Low

Low

High

High

Medium

High

Crosslinking methods

Ionically, thermal, pH mediated; polymerized, photopolymerized; enzymatic

Shear-thinning; ionically, thermal, pH mediated; polymerized, photopolymerized

Ionically, thermal; photopolymerized; enzymatic

Photopolymerized

Shear-thinning; ionically, thermal

Ionically, thermal

References

[20,22,34,35,44,102]

[27,47-50,65,66,103]

[16,70,71,104]

[75,76,94,95]

[86,90,94]

[32,101,105,106]

3. Bioprinting process regulating stem cell properties In bioprinting process, stem cells will experience a period of transfer from a reservoir to an arranged position, during which external factors, e.g. mechanical stress, heat, radiation may be involved. These factors will inevitably influence stem cell properties Therefore, a systematic review of the interplay between stem cells and bioprinting process is necessary to customize bioprinting techniques for stem cells with regulated performance in subsequent culture.

3.1. Mechanical stress Mechanical stress is requisite to drive the cells and bioinks to the platform in any bioprinting techniques, leading to deteriorating of cell properties. Although the biochemical process of 18


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cell death caused by external force is still unclear, excessive deformation of cells under internal strain is reported as a phenomenological reason 107. As for stem cells, differentiation can be impacted by stress as well, since mechanical force can act on mechanoreceptor integrins, pericellular tethers, focal adhesions ion channels, cadherins, connexins, and the plasma membrane’s lipid rafts 108.

Mechanical stress can be divided into normal stress and shear stress. Normal stress refers to the stress perpendicular to the cell surface when bioinks are extruded through bioprinting machine or landed on the substrate, while shear stress refers to the stress parallel to the cell surface when viscous fluid bioinks flow through the nozzle. In nozzle-free bioprinting techniques such as laser-assisted bioprinting or stereolithography-based bioprinting, merely normal stress affects cells in most cases, thus an open-pool ejection system is suggested from the point of reducing potential loss of cell viability 22. Shear stress is regarded as the major cause of cell injury and death in bioprinting techniques with nozzles, especially microextrusion-based bioprinting with viscous bioinks (Figure 3A). Higher shear stress is generated by either greater driving force to improve printing speed and efficiency 21,109-111 or smaller nozzle diameter to increase printing accuracy 112-114. Viscous bioinks may cause more cell death due to increased shear stress, yet they can facilitate the shape fidelity of 3D structure 22. Shear thinning bioinks are proposed as a solution to improve cell viability, since their viscosity decreases as shear rate increases 22,115. As reported by Thakur A. et al., hydrogels can be modified with nanosilicates to enhance the shear-thinning characteristic for the better viability of MSC after injection through a nozzle 116. Non-viscous bioinks that 19



solidify rapidly after leaving needles serve as another choice, which can be realized by UV-light photo-crosslinking 117 or ionically crosslinking via coaxial extrusion 118. Jason Burdick’s group invented an in situ photo-crosslinking approach for bioprinting low-viscous cell-laden methacrylated hyaluronic acid through a photopermeable capillary to achieve immediate crosslinking prior to deposition (Figure 3B) 117.

3.2. Temperature and radiation Besides mechanical stress, temperature and radiation are other physical cues affecting stem cell properties during bioprinting process. Compared with low temperature, cells are more sensitive to high temperature, which can be resulted from localized heating in thermal inkjet bioprinting or thermal energy of UV light in laser-assisted bioprinting (Figure 3C). Thus, the regulation of temperature in the whole bioprinting process is significant in the design of bioprinting techniques 120. It was reported that localized temperature up to 300oC did not exhibit a substantial impact on the viability of the neural cells 121. As for laser-assisted bioprinting, Yaakov N. et al. demonstrated using near-infrared wavelength (700–1000 nm) could overcome the exposure of excessive thermal energy onto the cell biolayer and overheating of the cell, since photons in the near-infrared lack the energy to generate free radicals and are not absorbed by DNA 120. But moreover, radiation can be more dangerous in laser-assisted bioprinting, stereolithography-based bioprinting or any photo-crosslinking bioprinting techniques (Figure 3D). Schiele N. R. et al. demonstrated that excessive absorption of radiation energy may lead to cell injury and DNA damage of pluripotent embryonal mouse carcinoma cells 122, so the irradiation parameter ought to be monitored 20


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carefully.

3.3. Duration of unfavorable conditions During the whole process of bioprinting, cells experience an unsatisfying environment, including insufficient culture media, harmful chemicals (e.g. crosslinking agent) and complex physical cues. Therefore, the duration of unfavorable conditions is supposed to be shortened as much as possible to increase cell viability. For example, a shorter nozzle is proposed in bioprinting to reduce the extrusion time, when cells experience shear force 15. Cui X. et al. recommend that the maximum heating duration of each cell in thermal inkjet bioprinting should be limited to 2 µs 44. On the other hand, the duration has to be long enough to satisfy some process requirements, such as sufficient crosslinking for the construction of stable structures. In addition, loss of cell viability can also result from increased stress when printed with high speed while pursuing short printing time. Therefore, the optimization of bioprinting duration should be considered comprehensively.

21



Figure 3. Regulation of stem cell properties by controlling mechanical stress, temperature, and radiation in bioprinting process. (A) Stress distribution in the needle during microextrusion-based bioprinting. Comparing with static status, stress in flowing bioinks can lead to cell death near the shell of the needle. Reproduced with permission from ref 109. Copyright 2016 Advanced Healthcare Materials. (B) A structure generated by bioprinting of low-viscous methacrylated hyaluronic acid. In situ crosslinking makes it feasible to bioprint low-viscous bioinks with high fidelity, which can decrease shear stress in microextrusion-based bioprinting. Reproduced with permission from ref 117. Copyright 2016 Advanced Materials. (C) Viability of Chinese hamster ovary cells after thermal inkjet bioprinting. Heat in thermal inkjet bioprinting caused cell death, which is related to cell concentration. Reproduced with permission from ref 44. Copyright 2010 Biotechnology & Bioengineering. (D) Spreading and viability of MSCs influenced by radiation in laser-assisted bioprinting. Reproduced with permission from ref 77. Copyright 2017 Scientific Reports.

4. Microenvironmental factors modulating stem cell functions in subsequent long-term culture After bioprinting, stem cell-laden structures will be cultured for further investigation. The microenvironment constructed by cell printing technology involves many aspects, including micro- and macro-structure, biophysical and biochemical properties of bioink and culture medium, and mass transfer, etc. These microenvironment factors would vary over time and interact with stem cells to modulate their viability, proliferation and differentiation.

4.1. Biophysical cues 22


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4.1.1. Stability and biodegradability In many cases, bioprinted constructs need to be cultured in long-term for downstream applications. One of the most important issues is the survival and preservation of functionality of the encapsulated cells within the hydrogel bioink over time 110, thus posing an essential demand for structure stability. Besides the collapse of the entire construct, Müller M. et al. found that micro breaks can also reduce cell viability because of the distortion of stress field. Structure stability can be attributed mainly to the intrinsic properties of bioink and specific culture condition. Native biomaterials are employed in bioprinting for their prominent biocompatibility, while most of them lacks long-term mechanical strength 58. Hence, multi-materials or inorganic additions are also investigated 123. As for synthetic biomaterials, crosslinking is considered as a reliable solution to improve stability, with various choices of crosslinking mechanisms and reagents 22,115. Service performance is also significant in practical application, especially in vivo research or transplantation. In vivo condition will accelerate the degradation of materials by enzyme activity or other chemical reactions, leading to the loss of filler volume 124. Wang X. F. et al. reported that a low-viscous bioink consisting of gelatin and alginate embedding ASCs exhibited irregular morphology and loss of structure in vivo after osteogenic differentiation in vitro, which resulted in insufficient volume to fill the gap and lose connection between printed cells and tissues around, reducing the efficiency of bone regeneration 125.

But on the other hand, the bioinks should be replaced by extracellular matrix (ECM) ultimately. The ideal bioink should demonstrate the change of mechanical property in the 23



whole lifespan of bioprinting practice. That is, to support delicate structure after bioprinting, to maintain shape fidelity during cell culture, and to degrade gradually with the rate matching tissue fusion and maturation. The degradation process is suggested to keep pace with the production of ECM 15 and the proliferation of stem cells 126. For example, Rutz A. L. et al. invented a series of tunable hydrogels by mixing gelatin, fibrinogen, PEG, reactive group-modified PEG, and peptide amphiphile under different ratios, to alter construct degradation in accordance with ECM production, improving the viability and function of human dermal fibroblasts 127.

4.1.2. Stiffness Besides mechanical stability, stiffness is also strongly related to cell behaviors in stem cell bioprinting. Attachment to the matrix is realized via integrin-containing focal adhesions, which links to stress fibers such as actin-myosin cytoskeleton inside cells. These fibers can pull the matrix, sense resistance and provide mechanical signals to regulate cell spreading, morphology and differentiation 128. It is reported that with the same culture medium, MSC differentiation can be induced merely by matrix stiffness to the lineage of neural (0.1–1 kPa), myogenic (8–17 kPa) or osteogenic (>34 kPa), where signaling mediators of mechanotransduction like TAZ and YAP sense matrix stiffness and regulate MSC differentiation 24,129. Duarte Campos D. F. et al. has utilized this finding in microextrusion-based bioprinting of MSC, and demonstrated enhanced differentiation towards osteogenic in soft collagen-rich bioink and towards adipogenic in stiff agarose-rich bioink 129 (Figure 4A). 24


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4.1.3. Surface features Since the reaction between stem cells and bioinks merely exists on the material surface where cells attach, stem cell functions can be regulated by changing the surface features. Besides chemical synthesis or coating to introduce ligands which are benefit to cell attachment, survival or inducing desired differentiation, surface topography in nano- and micro-scale has a significant effect on cell behavior such as cell growth, morphology 130, migration, proliferation 131, differentiation 25 and cytoskeletal assembly 15 as well (Figure 4B). However, most research is still at the stage of 2D surface patterning with micro-topography 132. Surface modification of metal can be realized by acid etching, hydrothermal treatment and micro-arc oxidation 130, while topographical surface on polymers can be generated by photolithography and micromolding 132. Pioneer techniques have been developed in surface topography of 3D structure as well, where Poellmann M. J. et al. fabricated microscale polyacrylamide (PAM) on a second hydrogel by electrohydrodynamic jet bioprinting, where the behavior of MC3T3-E1 preosteoblast cells was regulated by the size of PAM droplets 133. In the future, bioprinting with higher resolution holds the potential to produce constructs with delicate topographic features from micro-pores to nano-structures to optimize extracellular environment.

Nowadays, mechanical and topological features have been widely applied in regulating stem cell proliferation and differentiation. Actually, other physical signals can be utilized to modulate post-bioprinting culture as well. For example, electrical stimulation was reported to 25



enhance neurogenesis of neural stem progenitor cells, neurite extension and neuron maturation 134. In the future, the creation of a comprehensive physical field around printed structures can be considered to enhance stem cell function and maturation.

4.2. Biochemical cues 4.2.1. Bioactive bioinks Besides biocompatibility to ensure cell viability, traditional bioinks are mostly inertia to cell differentiation to avoid undesired fate. However, since bioink functions as the major component of stem cell niche, bioactive bioinks have been explored recently to facilitate stem cell proliferation 135 or induce differentiation 26. Most bioactive bioinks are native materials or the mixture of native and synthetic materials, as biospecific signals are presented in their protein structure 136,137 (Figure 4C). For example, decellularized extracellular matrix (dECM) has been widely used in stem cell bioprinting where cells encapsulated exhibit intrinsic morphology, high viability, better proliferation and differentiation in vivo 26,138,139. Pati F. et al. reported that the engraftment, survival and long-term function of adipose-derived stem cells (ASC) and turbinate-tissue derived stromal cells were improved due to the crucial cues from tissue-specific dECM bioinks. As for differentiation, cells were induced for adipogenic differentiation by adipose dECM, chondrogenic differentiation by cartilage dECM and cardiogenic differentiation by heart dECM 137. Polypeptide-DNA hydrogel, as another example of bioactive bioinks, were invented by Li C. et al. for bioprinting of AtT-20 cells. Composed of polypeptide and DNA, this material is biodegradable and programmable under physiological conditions of proteases and nucleases 140.

26


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4.2.2. Intercellular signals Besides the cues from bioinks, signals released by other cells also direct stem cell behavior. Intercellular signals can be divided into intracrine, autocrine, juxtacrine, paracrine, and endocrine signals based on the distance between signaling and responder cells. In stem cell bioprinting, juxtacrine signaling and paracrine signaling are more significant due to the size of printed constructs nowadays. As for the former, it has been proved that intercellular communication can be realized by molecule transfer through gap junction channels along the contact surface 141. Stem cells which tend to form aggregates are influenced deeply by cluster situation under this mechanism. The differentiated degree of MSC into osteogenic or adipogenic lineage is reported much higher in aggregated cells than in isolated cells 142. Besides, the size of stem cell aggregates can regulate differentiation fate 14. With the control of the initial cell seeding density, bioprinting volume and culture time, stem cell aggregates can be formed with a regular shape and uniform size through cell proliferation after bioprinting 143. Ouyang L. et al. developed a method for mass production of embryoid bodies through bioprinting technology, where ESC pluripotency was maintained with optimized aggregate size and morphology 144. Contact intercellular signals also exist in cell aggregate bioprinting where aggregates are fabricated in advance, as Wang Z. et al. reported enhanced functionalization of MSC aggregates in printed gelatin-based structure, comparing with single MSCs under the same bioprinting condition 145.

Paracrine signals are released by signaling cells and target the cells in the vicinity. One of the most important employment is co-culture of multiple cells to improve cell functions. Roy N. 27



S. et al. reported that differentiation of human ESCs towards dopaminergic neuronal fate can be enhanced by co-culture with human fetal midbrain astrocytes 146. Yao R. et al. found co-culture of ASC and human umbilical vein endothelial cells (HUVEC) encapsulated in collagen/alginate microsphere could facilitate in vivo vascularization and formation of adipose tissues 147. Co-printing of different types of cells to control cell migration has been studied as well, where Bourget J. M. et al. printed HUVECs and BMSCs together by laser-assisted bioprinting to avoid HUVECs spreading out of the printed collagen structure 148. What’s more, deposition of multiple cells in arranged postion can facilitate construction of complex tissues. For example, co-printing of four bioinks including poly(dimethyl siloxane), Pluronic F127 (PF127), GelMA encapsulating human neonatal dermal fibroblasts (hNDF), and GelMA encapsulating 10T/12 fibroblasts, is developed by Kolesky D. B. et al. to fabricate vascularized, heterogeneous tissue constructs 149 (Figure 4D). Besides, paracrine signaling can be regulated by changing cell status, subsequently affecting cell functions. For example, Snyder J. et al. reported that stem cells with ruptured membrane caused by elastic and inelastic strain in bioprinting will release necrotic distress signals, which could initiate migratory, biochemical synthesis and differentiation behavior in MSCs 119. In addition, control of cell density and viability impacts the local concentration of bioactive proteins, further influencing stem cell differentiation. 4.2.3. Additional factors Additional factors involved in bioengineering studies include cytokines, hormones, and enzymes. Adding cocktail of factors for stem cell induction is considered as a proven technique in 2D stem cell differentiation, which has been widely utilized in 3D differentiation 28


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as well. One method is to supplement these chemicals to culture media immersing the printed structure, similar to 2D differentiation. However, bioinks may hinder the transfer of additional factors, as the cells are encapsulated in bioinks but not exposed directly to the solution. Sometimes, this feature may lead to a worse outcome compared with 2D differentiation 150. Hence, another method is proposed to immobilize the additional factors in bioinks, where the better dispersion of these factors and interactions with stem cells can be achieved 151,152 (Figure 4E). Besides, Poldervaart M. T. et al. explored improved osteogenesis differentiation of goat multipotent stromal cells by incorporation of bone morphogenetic protein-2 (BMP-2) into bioinks 153. Since multi-factors can be printed into different places precisely with pre-designed patterns, complex tissues can be achieved by regulating growth and differentiation of cells in different position with different factors. Ker E. D. et al. bioprinted BMP-2 and fibroblast growth factor-2 (FGF-2) with spatial difference to induce C2C12 myoblasts to osteoblasts and tendon cells respectively to generate musculoskeletal models 154. Apart from directing differentiation, additional factors show other functions, such as improvement of cell adhesion, migration and proliferation 126. Moreover, adding biodegradable units in bioinks will allow adjustable degradation rate of materials to match better with ECM production 155. West J. L. et al. found that incorporation of enzymatically degradable oligopeptide sequences in PEG can modulate degradation rate since local enzymes will recognize and function at enzymatic cleavage sites within the bioink, resulting in the optimization of cell migration and tissue formation 156.

4.3. Mass transfer 29



4.3.1. Porosity Bioinks show the porous property as the basic approach of mass transfer inside the construct. It is involved in the essential requirement of biocompatibility to provide space for cell spreading and migration, and enable the exchange of chemicals to facilitate cell growth 125,157 (Figure 4F). As for stem cells, factors dissolved in culture media need to be transferred to induce differentiation, So the number, shape and size of micropores, makes a difference in the efficiency of stem cell proliferation and differentiation 150,158. It was reported by Domingos M. et al. that MSC attachment and migration was associated with the pore size and shape of bioprinted PCL scaffold. These feature also affected the subsequent vascularization for further mass transfer 159. It should be mentioned that only open pores can facilitate the cell function, as they enable exchange of various chemicals inside and outside the bioinks, and access of encapsulated cells to the implant are feasible through these pores or channels 160.

But on the other hand there is a negative effect of porosity on structure stability, which can be aggravated when inner non-connected molecules are eluted off. For example, Duarte Campos D. F. et al. found that agarose-chitosan blends, which has better porosity for cell migration were unprintable due to unsatisfactory stability, unlike agarose and agarose-collagen hydrogels which are widely used in bioprinting 135.

4.3.2. Vascularization Porous bioinks ensure the mass transfer in the early stage when cells are evenly distributed in the structure. However, with the degradation of bioinks and the maturation of printed tissue, 30


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close-packed cells would impede the penetration of solution. It is reported that solid tissues with the thickness over 200 µm have limited clinical application 161 and cells can hardly survive in tissues with thickness over 1mm 162, which can be more serious for highly vascularized tissues such as liver, kidney, spleen, etc 163. Vascularization has become one of the bottleneck problems in the application of engineered 3D tissues with clinical-relevant dimension 164.

With the help of 3D bioprinting technology, angiogenesis elements can be constructed with desired patterns to facilitate vascularization 165 (Figure 4G). Co-printing vessels within tissues are mostly employed for the construction of vessels with large diameter (>6mm) like aortas. Direct bioprinting of fine vessel structure is inefficient under current techniques with inadequate resolution, especially for capillaries, hindering fabrication of mature vascularized tissues 166. Considering the complex structure of nature vessels, temporary supporting materials is printed and then removed to assist the formation of hollow channels. In addition, bioprinting of aggregates facilitates the construction of vessels of large scale 92.

Autogenous vascularization methodology focuses more on the potentials of stem cells rather than bioprinting process. Stem cells with differentiation potential into vascular tissues, including endothelial progenitor cells (EPC) 167 and MSC 168, have been utilized in the bioprinting vascularized structure under proper induction. But the progress of either vasculogenesis or angiogenesis is time-consuming, where the viability of complex organs may decrease sharply before the completion of this progress 16. Therefore, Mironov V. et al. 31



proposed a hybrid method by co-printing of cell aggregates for fabrication of branching large vessels and pre-vascularized tissue building blocks as a microvascular network, to form a vascular network with different sizes and types of vessels for organ bioprinting 97. Moreover, attention should be paid to the connection between printed vascular channels and host vasculature 166.

Figure 4. Microenvironmental factors modulate stem cell functions in long-term culture. (A) Bioink stiffness affects osteogenic differentiation of MSC, stained by oil red O. Reproduced with permission from ref 129. Copyright 2015 PloS one. (B) Fabricate surface nano-features to regulate cell morphology and migration. Reproduced with permission from ref 130. Copyright 2016 Colloids and 32


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Surfaces B: Biointerfaces. (C) Bioprinting hybrid bioactive structure of ECM to improve biocompatibility and PCL as the framework. Reproduced with permission from ref 137. Copyright 2014 Nature Communications. (D) Co-printing of 10T1/2 fibroblasts (blue), hNDFs (green), HUVECs (red) can facilitate the formation of heterogeneous tissue constructs by paracrine. Reproduced with permission from ref 149. Copyright 2014 Advanced Materials. (E) Tethering RGD ligands onto ɑ-cyclodextrin-modified PEG can enhance spreading of MSCs. Reproduced with permission from ref 152. Copyright 2016 Advanced Materials. (F) Porous internal structure of alginate/gelatin hydrogel is essential for mass transfer and cell migration. Reproduced with permission from ref 125. Copyright 2016 PloS one. (G) HUVECs (red) printed in hNDF-laden (green) matrix form a linear vessel with the lumen to support perfusion for tissue mature in long-term culture. Reproduced with permission from ref 165. Copyright 2016 Proceedings of the National Academy of Sciences.

5. Bioinks regulating printability and stem cell functions Bioinks are considered a vital element in stem cell bioprinting technology, since they function as one of the bioprinting elements, delivery vehicle of stem cells and also a very important aspect of microenvironment for subsequent culture. In this section, we elaborate the requirements and rationale of bioink properties from the perspective of bioprinting process and stem cells, respectively. Some literature discussing bioink materials can be refereed to for more details 169,170.

5.1. Printability: requirements of bioprinting process The status of bioinks can be briefly divided into three stages throughout the whole bioprinting 33



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process: 1) before bioprinting: liquid/sol state ; 2) during bioprinting: rapid solidification and 3) after bioprinting: solid/gel state. A printable bioink ought to display different properties in accordance with the demand of different stage.

Flowability is essential to bioinks in the first stage, not only for encapsulating stem cells expediently but for providing more options of bioprinting parameters as well, like printing speed. As higher viscosity and higher printing speed can both generate greater mechanical forces that pose harm to stem cells, viscous bioinks limit the printing speed to maintain cell survival

65,123

. In addition, different enabling bioprinting techniques show the distinguished

optimal range of bioink viscosity, where most bioinks with viscosity over 300 mPa·s can merely be applied in microextrusion-based bioprinting 10.

In the second stage, to establish a pre-designed structure with accuracy, the ability of rapid solidification under the mild condition is prerequisite for bioink properties, which is realized by crosslinking in most cases. Ideally, crosslinking of bioinks is supposed to take place upon reaching the substrate and no sooner than extrusion from printing head (inkjet and microextrusion-based bioprinting) or printing ribbon (laser-assisted bioprinting)

25

.

Meanwhile, the crosslinking condition should cause minimal cell injury or death. Generally, crosslinking methods can be divided into physical crosslinking, chemical crosslinking and enzymatic crosslinking. Physical crosslinking does not involve the formation of covalent bonds and is reversible for most bioinks. Its mechanism includes ionic reaction, complexation reaction and hydrogen bonding reaction, which often occurs under the variation of charges, 34


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temperature, and pH-value. By polymerization, chemical crosslinking is irreversible and can create more stable structures under covalent reaction. As one of the most significant methods in chemical crosslinking, photo-crosslinking has been widely employed and is the main approach for stereolithography-based bioprinting. Under irradiation, photo-initiators in bioinks can generate excited molecules to activate polymerization of monomers and oligomers

68

. Enzymatic crosslinking happens when corresponding protease and suitable

conditions are provided. For instance, fibrinogen can be converted by thrombin into fibrin, which is a typical process in blood coagulation 39.

In the last stage, printed structures are required to maintain the shape through the subsequent cell culture period. Crosslinked bioinks should provide long-term stability in the culture medium, while sustaining stem cells proliferation, migration, metabolism and morphogenesis. In addition, swelling and/or shrinking of bioinks may influence the integrity, size and resolution of printed structures

123

. These factors must be taken into account in the design of

printing process. However, the addition of stem cell in the bioinks may also affect fluid and crosslinking properties. Therefore, a simple test of bioink physical properties without cells is inadequate, and multiple experiments are necessary to optimize and standardize the bioink properties and printing process.

5.2. Biocompatibility: requirements of stem cells During bioprinting, stem cells are evenly distributed in bioinks. After bioprinting, crosslinked bioinks provide a long-term microenvironment for stem cell culture. Therefore, interactions 35



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between stem cells and extracellular bioinks have a significant influence on stem cell viability, attachment 15, proliferation 135, differentiation 21, migration 126 and morphogenesis 171.

Stem cells can be harmed by mechanical stress during bioprinting process, restricting the use of viscous bioinks 22. Meanwhile, the toxicity of all the chemicals involved in bioprinting and subsequent culture should be checked to avoid cell death, including bioinks, additions like photo initiators 172, by-products during crosslinking 123, and degradation products 173.

Stem cell attachment, migration and morphology are mainly impacted in post-printing culture. Ligands between stem cells and polymer chains determine whether cells can attach to the surface of bioinks facilely or detach for migration 159. What’s more, bioinks with high strength and stiffness are disadvantageous for cell mobility. Thus chemical crosslinked bioinks usually demonstrate less biocompatibility than physical crosslinked ones since they have higher

stability

152

. A common method to tune cell attachment, migration and formation of specific

morphology such as cell aggregations without changing the matrix of structure is surface modification, including generation of nano-scale topographies

131

or bonding of chemical

ligands 152.

Provided with 3D microenvironment, stem cells exhibit better proliferation in printed structure compared with 2D culture 144. Increased proliferation can be achieved by optimizing alignment of bioinks for some stem cells like MSCs

135

. Porosity is another factor regulating

stem cell proliferation by controlling the transfer of oxygen, nutrition and metabolite 36


126

. In

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addition, the degradation rate of bioinks should be in accordance with proliferation rate of stem cells and production of stem cell ECM, in order to offer adequate space for cell growth and tissue maturation 15.

Distinct from bioprinting of primary cells, differentiation is one of the most important feature of stem cells. On the one hand, bioinks should not result in undesired differentiation, which may occur in dECM and some other natural bioinks. On the other hand, stem cell differentiation can be modulated by change of physical properties or chemical components. Thus, a comprehensive optimization of bioinks is valuable for improved stem cell differentiation.

In all, the choice of bioinks involves a delicate balance between printability and biocompatibility. Generally, natural bioinks offers better support for stem cell functions, but usually shows poorer mechanical properties and repeatability, probably due to batch-to-batch variations. Artificial bioinks are more stable and controllable, but demonstrate less biocompatible in most cases. Most commonly used bioinks for stem cell bioprinting are listed in Table 2.

37



Table 2. Commonly used bioinks in stem cell bioprinting

38


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Bioprinting technique

Crosslinking method

Cell type

Viability(%)

Ref.

Agarose

Micrextrusion-based bioprinting

Thermal

MSCs

98.8

135

Agarose/alginate/chitosan


Ionically

NSCs

>75

62

Alginate


Ionically

MSCs

95

46

Inkjet bioprinting

Ionically

ESCs, iPSCs

90

21


Ionically

ASCs

91

54


Ionically

ASCs, preosteoblasts

92.3

174


Ionically

MSCs

100

71


Thermal, ionically

ESCs

90

27


Ionically

Glioma stem cells

86.9

63


Ionically

MSCs

89

125

Chitosan


pH mediated

ASCs

-

175

Collagen type I

Inkjet bioprinting

pH mediated

MSCs

81

102

Collagen type I/fibrin

Inkjet bioprinting

pH mediated, enzymatic

NSCs

92.9

19


Enzymatic

Amniotic fluid-derived stem cells

-

104

Fibrin

Inkjet bioprinting

Enzymatic

MSCs

-

102

Gelatin

inkjet bioprinting

Thermal, polymerized

MSCs

-

102


Thermal

ESCs

-

73

Inkjet bioprinting

Photopolymerized

MSCs

90

18


Photopolymerized

BMSCs

-

77


Photopolymerized

NSCs

-

82

GelMa/PEG

Inkjet bioprinting

Photopolymerized

MSCs

>80

39

Hyaluronic acid/matrigel


Polymerized

MSCs

-

176

PEG


Photopolymerized

MSCs

90

177

polyurethane


Thermal

NSCs

>50

61

Alginate/Collagen type I

Alginate/gelatin

GelMa

6. Perspective 39



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Stem cell bioprinting takes advantage of both the precise positioning of the substance of bioprinting technology and the proliferation and differentiation abilities of stem cells, showing great potential in stem cell biology, drug testing, tissue engineering and regenerative medicine research. But this emerging technology faces many challenges related to cellular, technical, and material aspects in the whole lifespan. In recent years, significant progress has been achieved in improving stem cell functions and creating more complex structures.

Creation of microenvironment comparable to natural tissue/organ is proposed as a direction to improve stem cell behaviors in printed structure. On the one hand, the invention of novel printable biomimetic materials is of significant importance. Composite materials can be utilized as bioinks combining the printability of synthetic materials and biocompatibility of natural materials. Besides, functional bioinks can be also generated by extra additions or surface modification, which ensure better proliferation, induced differentiation and intelligent degradation

173

. On the other hand, precise spatial distribution of multiple bioinks with

suitable mechanical properties and other chemicals in accordance with the stem cell demands can help regulate microenvironment in micro-scale. Merceron T. K. et al. reported that a muscle-tendon unit construct was fabricated by bioprinting four bioinks encapsulated with myoblasts and fibroblasts with regionally defined characteristics

178

. In addition, to predict

cell behaviors in the microenvironment, computer modeling has been utilized in the research of post-printing cell culture, including kinetic Monte Carlo (KMC) simulations particle dynamics (CPD)

180

179

, cellular

and phase field theories 96. Currently, these modeling techniques

are mainly focused on cell aggregate fusion or stress distribution inside the structures. 40


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Simulation of flow velocity, stress, and temperature distribution in the fluid to predict stem cell status in bioinks during bioprinting is necessary. Further development in the modeling of biochemical environment around stem cells after bioprinting is more desired. In the future, modeling of stem cell situation in bioinks from loading to long-term culture is needed.

Another significant direction is scale-up and mass production for clinical and/or commercial application. On one hand, high throughput technology is prerequisite for mass production with quality controllability. On the other hand, commercial availability and practicability of stem cells and bioinks should be also considered, including source, cost, shelf life and FDA administration. In addition, equipment and technologies should be appropriate for scale production. Before bioprinting, stem cells are supposed to proliferate in controlled ways to provide adequate cell numbers for bioprinting. Pre-differentiation of stem cells and pre-fabrication of cell aggregates in quantity are sometimes required in specific research. After bioprinting, structures with stem cells are proposed to be cultured in bioreactors for fusion of cells and maturation of tissues. A further step is the establishment of assembly line for organ biofabrication, whose components involve clinical cell sorters, stem cell propagation bioreactors, cell differentiators, cell aggregate biofabricators, cell encapsulators, robotic bioprinters, and perfusion bioreactors. The whole assembly line should be automatic, biocompatible, large-scale, and flexible for generation of diverse tissues and organs

181

. A

real-time monitor of cell status is suggested to inform researchers and regulate fabrication process. What’s more, since most laboratories around the world utilize home-made or customized 3D bioprinters for specific research nowadays, standardization is another critical 41



step in the industrialization of stem cell bioprinting

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. Tissues and organs should be

generated by the standardized process in a reproducible way.

In the aspect of clinical application, stem cell bioprinting shows great potential in precision medicine. Stem cells are obtained directly from a patient or modified with individual genetic content. By bioprinting and subsequent differentiation, specific tissues or organs are available as either in vitro testing models or in vivo implants for diagnosis, analysis, and treatment of personal disease. In recent years, bioprinting of iPSCs offers a potential versatile approach for production of customized tissues, as this type of cells can be acquired from every patient by reprogramming of adult cells, and differentiate into desired cell types 21. Though challenges still exist in the functional stability and security of iPSCs and impact of bioprinting process on iPSCs, but the application of iPSC bioprinting hold a bright future for precision medicine.

In conclusion, even still in early stage, 3D bioprinting of stem cells is emerging as a promising technology which has potential applications in many research. Bioprinting technology provides a convenient and repeatable approach for precise spatial positioning of both cells and bioink, which would facilitate the delicate study of cell niche regulation, which is hardly feasible in other 3D culture methods. Meanwhile, stem cells serve as the reliable cell sources in bioprinting for tissue engineering, regenerative medicine, drug testing and cancer studies. Their proliferation and self-renewal property facilitates rapid fabrication of large-scale tissues or even organs on demand such as healing of large skin wounds

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addition, induced differentiation of stem cells in bioprinted structure can be combined with 42


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organoid technology and work as a versatile tool for construction of complex functional tissues of various types. With enhanced understanding of the interplay between stem cells and microenvironment and improvement of bioprinting process, bioinks and stem cell technology, future application of 3D bioprinting of stem cells can be extended to drug development, organ transplantation and personalized medicine as a reliable, convenient and efficient technology.

Acknowledgements The authors acknowledge fundings from the National Natural Science Foundation of China (No. 31500818 and No. 31711530218).

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For Table of Contents Use Only

Manuscript title: of bioprinting

"Bioprinting of

stem cells: interplay

process, bioinks and stem cell

proper

Supeng; Feng,

Zhu,

ties."

Author(s): Ding, Fei; Tan, Ze'en;

Lu; Wu, Jiayang;

Yao, Rui

69



Figure 1. Schematic of the whole lifespan of stem cell bioprinting practice. Reproduced with permission from ref 27,28. Copyright Biofabrication. 1763x1234mm (72 x 72 DPI)


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Figure 2. Illustrations and examples of different bioprinting techniques. (A) Multi-cell bioprinting, which can be further divided into inkjet bioprinting (image from MicroFab Technologies, Inc.), microextrusion-based bioprinting (image from Organovo Holdings, Inc.), laser-assisted bioprinting (image from Organovo Holdings, Inc.) and stereolithography-based bioprinting (image from Formlabs, Inc.). (B) Cell aggregate bioprinting. Reproduced with permission from ref 30. Copyright Biomaterials. (C) Single-cell bioprinting. Reproduced with permission from ref 31. Copyright Biomaterials. 1336x1871mm (95 x 95 DPI)



Figure 3. Regulation of stem cell properties by controlling mechanical stress, temperature, and radiation in bioprinting process. (A) Stress distribution in the needle during microextrusion-based bioprinting. Comparing with static status, stress in flowing bioinks can lead to cell death near the shell of the needle. Reproduced with permission from ref 109. Copyright Advanced Healthcare Materials. (B) A structure generated by bioprinting of low-viscous methacrylated hyaluronic acid. In situ crosslinking makes it feasible to bioprint low-viscous bioinks with high fidelity, which can decrease shear stress in microextrusion-based bioprinting. Reproduced with permission from ref 117. Copyright Advanced Materials. (C) Viability of Chinese hamster ovary cells after thermal inkjet bioprinting. Heat in thermal inkjet bioprinting caused cell death, which is related to cell concentration. Reproduced with permission from ref 44. Copyright Biotechnology & Bioengineering. (D) Spreading and viability of MSCs influenced by radiation in laser-assisted bioprinting. Reproduced with permission from ref 77. Copyright Scientific Reports. 169x110mm (300 x 300 DPI)


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Figure 4. Microenvironmental factors modulate stem cell functions in long-term culture. (A) Bioink stiffness affects osteogenic differentiation of MSC, stained by oil red O. Reproduced with permission from ref 129. Copyright PloS one. (B) Fabricate surface nano-features to regulate cell morphology and migration. Reproduced with permission from ref 130. Copyright Colloids and Surfaces B: Biointerfaces. (C) Bioprinting hybrid bioactive structure of ECM to improve biocompatibility and PCL as the framework. Reproduced with permission from ref 137. Copyright Nature Communications. (D) Co-printing of 10T1/2 fibroblasts (blue), hNDFs (green), HUVECs (red) can facilitate the formation of heterogeneous tissue constructs by paracrine. Reproduced with permission from ref 149. Copyright Advanced Materials. (E) Tethering RGD ligands onto ɑcyclodextrin-modified PEG can enhance spreading of MSCs. Reproduced with permission from ref 152. Copyright Advanced Materials. (F) Porous internal structure of alginate/gelatin hydrogel is essential for mass transfer and cell migration. Reproduced with permission from ref 125. Copyright PloS one. (G) HUVECs (red) printed in hNDF-laden (green) matrix form a linear vessel with the lumen to support perfusion for tissue mature in long-term culture. Reproduced with permission from ref 165. Copyright Proceedings of the National Academy of Sciences. 162x162mm (300 x 300 DPI)



Table 1. Comparison of bioprinting techniques 385x193mm (96 x 96 DPI)


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Table 2. Commonly used bioinks in stem cell bioprinting 529x776mm (72 x 72 DPI)


Bioprinting of Stem Cells: Interplay of Bioprinting Process, Bioinks, and

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