Bioreactor Synergy with 3D Scaffolds: New Era for Stem Cells Culture

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Bioreactor synergy with 3D scaffolds: new era for stem cells culture Tianqi Yi, Shaoxiong Huang, Guiting Liu, Tiancheng Li, Yang Kang, Yuxi Luo, and Jun Wu ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00057 • Publication Date (Web): 10 Jul 2018 Downloaded from http://pubs.acs.org on July 17, 2018

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ACS Applied Bio Materials

Bioreactor synergy with 3D scaffolds: new era for stem cells culture

Tianqi Yi a‡, Shaoxiong Huang a‡, Guiting Liu a‡, Tiancheng Li a, Yang Kang b*, Yuxi Luo a*, Jun Wu a, c*

AUTHOR ADDRESS a

Key Laboratory of Sensing Technology and Biomedical Instrument of Guangdong

Province, School of Biomedical Engineering, Sun Yat-sen University, Guangzhou 510006, China. b

Key Laboratory of Mountain Ecological Restoration and Bioresource Utilization,

Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China. c

Key Laboratory of Polymer Composites and Functional Materials of Ministry of

Education, Sun Yat-sen University.

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KEYWORDS 3D scaffolds; Bioreactor; Microenvironments; Stem cell; Niche; Smart control.

ABSTRACT

Because of unparalleled advantages over other cells, stem cells are widely used in genetic diagnosis, drug delivery, and regenerative medicine. However, due to the content of stem cells in the organism is far from satisfactory, it is of great significance of stem cells to in vitro proliferation and differentiation. However, many stem cell cultures have low expansion efficiency and stem cells lose their value-adding ability and differentiation ability after many generations of culture. In order to solve these problems, people hope to more realistically simulate the micro-environment in which stem cells grow in vivo. Cell scaffolds gradually evolve from 2D structures to 3D structures. Addition of growth factors influences cell behavior from internal biochemical conditions, and the development of smart bioreactors gradually make progress, to more precise regulate the external conditions of stem cell. In this paper, the key factors for constructing the microenvironment of stem cell growth were analyzed and we reviewed the application of bioreactors and three-dimensional scaffolds in stem cell cultivation. Finally, this paper indicated the development directions of stem cell culture in vitro.


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1. Introduction

Stem cells are known for the capability of differentiate, regenerate and evolve to multiple lineages of cell types.1 In clinical treatment, a large number of stem cells are urgently needed as it is an ideal tool for transplantation or repairmen of damaged tissue in some congenital or acquired diseases treatment.2-3 For example, in a tissue replacement of myocardial infarction, 1×109-2×109 cells are needed and at least 1.3×109 β-islet cells are transferred to the cell during Islet transplantation.4-5 However, even in the bone marrow which is relatively abundant in stem cells, there is an average of one hematopoietic stem cell per 10,000 cells.6 Due to the great need of stem cells, it is essential to culture stem cells in vitro until it expands and differentiates to a certain amount for treatment. At present, most laboratories use 2D culture, the expand efficiency of which is still not ideal. Moreover, after a long-term cultivation, stem cells will not only lose the ability to proliferate, but also lose the ability of multi-directional differentiation.

The key to better growth of stem cells is to create a suitable microenvironment. The cellar microenvironment refers to the area where cells grow and metabolize. The role of the microenvironment is mainly reflected in three aspects. The three-dimensional structure affects the direction of cell growth and movement and ultimately determines the shape of the tissue. The biochemical internal conditions, which determine the cell adhesion and migration, contains growth factor that determines the cell proliferation and differentiation behavior. The external stimulus conditions, including mechanical 3 ACS Paragon Plus Environment


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factors, dissolved oxygen, pH, etc., affect the level of cell metabolism and cell differentiation to some extent.

For better create cellular microenvironments, some techniques have been used in cell cultivation (Figure 1). For the external microenvironment, the bioreactor could provide a uniform culture environment by means of dynamic culture. It has become a hot spot for providing external stimuli for cells while monitoring microenvironment parameters in real time. For the internal microenvironments, the three-dimensional tissue engineering scaffold has also been well applied in achieving cell high density growth and regulating cell growth and differentiation. It must be noted that the combination of bioreactors and three-dimensional tissue engineering scaffolds could more accurately simulate the growth environment of stem cells in the human body and achieve good results in cell culture. In this paper, we analyzed the key factors for constructing the microenvironment of stem cell growth and reviewed the application of bioreactors and three-dimensional scaffolds in stem cell cultivation. Finally, this paper indicated the development directions of stem cell culture in vitro.

2. Stem cells and microenvironments for proliferation and differentiation of stem cells

Adult Stem Cells (mainly Mesenchymal Stem Cells) are widely distributed in human tissue, which can self-renew and differentiate into other cells such as osteoblasts, adipocytes, chondrocytes, etc.7 MSCs was found in the bone marrow at first and it can also be found in other places when stimulated by specific signals.8 Because of its 4 ACS Paragon Plus Environment

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therapeutic role by producing blood cells to promote organ homeostasis, wound healing and characterization of easy preparation (easily isolated and amplified from the bone marrow) without promoting rejection, MSCs have become a widely accepted and extremely outstanding cell source. This report is mainly focused on MSC cultures.

2.1 History and backgrounds

In 1970s, Friedenstein was the first one to reveal Mesenchymal stem cell nature. Consequently MSCs were identified and isolated from bone marrow. Current research has shown that MSCs could also be isolated from cord cells, adipose tissue, molar cells, and amniotic fluid etc. Currently, bone marrow is still the indispensable source of MSCs. Frieden stein also proved MSCs role in keeping the hematopoietic niche.9 In the 1980s, MSCs were shown to differentiate into many other kinds of cells.10-11 Whereas, in the 1990s, MSCs were shown to keep their differentiation ability when cultured in vitro.12 During early 21st century, MSCs were seen to inhibit T-lymphocyte proliferation , thus identifying the possibility of allogeneic transplantation and immunomodulatory.

Morphologically seen from a microscope, MSCs are thin and long. They contain a large, round nucleus. In fact, a cell showing plastic adherent properties under normal culture conditions and has a fibroblast-like morphology can be classified as an MSC, according to standards proposed by the International Society for Cellular Therapy (ISCT). Besides, some surface markers such as CD73, CD90 can be expressed by 5 ACS Paragon Plus Environment


MSCs but some such as CD11b, CD14 are what MSCs lack. One of the most vital influences of MSCs is that they help keeping the balance of stem cells niche, where stem cells proliferation and differentiation are monitored and controlled.13 Furthermore, MSCs are often used to reconstruct niche.14

2.2 Application

Cellular therapies include administration of functional and healthy cells to replace dysfunctional cells damaged or destroyed by disease. MSCs are most promising candidates for use as potential therapeutic agents in cellular therapies and tissue engineering (Figure 2) because of MSCs wider distribution, easier separation and proliferation in vitro and higher maintenance of multipotency compared to other stem cells. The most encouraging characteristic of MSCs are their suitability for use in allogeneic transplantation as well as autologous transplantation since MSCs appear to be immunologically unique and won’t cause immune response and possible rejection.15-16

2.2.1 Facilitate Tissue Repair

MSC releases a large number of immunomodulatory and trophic bioactive factors at the site of injury. The trophic activity can prevent cell apoptosis and scarring and also stimulate the production of endogenous progenitor cells and angiogenesis while immunomodulatory mainly plays an important role in the clinical application of heterologous stem cells to prevent autoimmunity.17 Pericytes, a generalized MSCs located in the wall of the vasculature at the basement membrane, is the predecessor of 6 ACS Paragon Plus Environment

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precursor cells having functions to repair tissue and maintain normal physiological activities. During the event of tissue injury, pericytes lose contact with basement membrane and endothelial cells, start intense proliferation and migrate to adjacent damaged areas and divide and secrete several bioactive factors that function to protect and repair or regenerate the damaged tissue. Immune surveillance can be controlled at a low level by those bioactive factors to inhibit T and B cells from damaging tissues. Trophic factors secreted by the MSCs also exert immunosuppressive effect on the surrounding tissue that leads to minimal inflammatory response, reduced apoptosis, inhibit

scarring,

stimulate

angiogenesis

and

carcinogenesis

and

induce

chemoattractant effect on other cells. This also leads to stimulation of tissue-intrinsic progenitor to regenerate the damaged tissue and help in remodeling tissue.18-19

One of the most obvious and realistically achievable clinical application of MSCs currently is as a means of replacing damaged bone and osseous defects in bone tissue engineering. A major clinical goal in the treatment of osseous defects to develop new cellular therapies and tissue engineering strategies is becoming an increasing impetus in the future as improved quality of life leads to an increased life expectancy and consequently a higher incidence of age-related disorders such as osteoporosis. A number of factors can contribute towards the development of osseous defects and bone trauma, such as fractures, tumor invasion or genetic disorders. Current treatment strategies consist predominantly of autologous bone grafts and to a lesser extent allogeneic bone grafts, metallic implants and bio-ceramics. Unfortunately, as is the case for adipose tissue defects, the success of these current treatment methods is 7 ACS Paragon Plus Environment


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somewhat inadequate, for limited success including limited autograft supply/volume, infection due to pathogen transfer /immunological rejection following allografts, loss of function/donor site morbidity and, in some cases, even nerve damage. It is therefore hardly surprising that alternative strategies in the treatment of osseous defects are currently sought, with a particular clinical goal being the development of bone tissue engineering technologies to replace tissue damaged as a result of disease. MSCs are therefore highly appealing candidates for use in the cellular treatment of osseous defects due to their intrinsic osteogenic ability upon appropriate stimulation.

2.2.2 Regenerative medicine

Regenerative medicine refers not only to the repair, replacement and regeneration of defective organs, but also to all the endogenous and exogenous repair and regeneration of damaged tissues caused by trauma, disease and aging. The application of stem cell therapy in regenerative medicine presents a promising prospect for new biologic therapies. MSCs can avoid the ethics and tumorigenicity problems and have extensive sources that can be obtained in umbilical cord, placenta, endometrium, fat, deciduous teeth, bone marrow. What’s more, MSCs from different sources show different characteristics, such as MSCs from fat have the better effect in the treatment of ocular fundus diseases due to its strong ability to secrete cytokines. The ability of umbilical cord MSCs to propagate is superior to that of fat and bone marrow. The advantage of MSCs is also reflected in their multidirectional differentiation. Generally considered in the process of treatment, alternative differentiation into the new 8 ACS Paragon Plus Environment

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organization is the main treatment mechanism, but in recent years, many studies have shown that immunomodulatory and paracrine effects regulated by various biological factors including growth factors, cytokines, chemokines secreted by MSCs play a vital role in the growth and repair of tissues and organs. The stem cell therapy has the potential to be used in skin damage, mental derangement rational pain and ovarian premature aging, retinal pathological changes and autoimmune diseases such as rheumatoid arthritis.20-21

2.2.3 Therapeutic Potential in the Nervous System

Neurological disorders result from disruption in the structure and/or function of nervous tissue, either in the central nervous system, peripheral nervous system, or both.22-23 Neurodegenerative disorders are a further sub-classification of neurological disorders, which describes disorders that result from a loss and/or degeneration of nervous tissue. Current therapies for the treatment of neurological and neurodegenerative disorders are largely ineffective and inadequate. The development of effective therapies in this area is further complicated owing to the significantly varying pathologies between different disorders and even between individuals suffering from the same disorder. Consequently, the development of standardized therapy in the treatment of such conditions has not been achieved, as patients require treatments that are tailored specifically to their unique clinical symptoms. With the major development in stem cell research over recent years, there is the potential of cellular therapy involving the administration of stem cells and/or their derivatives to 9 ACS Paragon Plus Environment


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replace damaged and/or degenerated nervous tissue. With evidence to suggest that MSCs display greater plasticity than previously anticipated, concomitant with unique immunological properties which make them promising for both autologous and allogeneic transplantation. Thus, MSCs might present themselves as promising candidates in the treatment of neurological and neurodegenerative deficits.

2.3 Culture

2.3.1 MSCs Niche In 1978, Schoefield first put forward the item “niches”.24 He thought stem cells niches were definitive of anatomical location. When removed from niches, stem cells would differentiate. Generally speaking, stem cell niches are defined as the in vivo or in vitro microenvironment where stem cells settle and are stimulated by signals. Niches cannot be seen as some specific physical position alone, but some position where exogenous signals interact, and finally cause impact on behaviors of stem cells. There signals include interactions between stem cells, cells, the extracellular matrix and some factors which can activate or suppress stem cells behaviors. From the results, stem cells might be able to keep their stemness, self-renew or differentiate into other kinds of cells.

2.3.1.1 MSCs niches and culture requirements

In fact, the limited number of MSCs in vivo limits their application, thus making it prerequisite to scale up MSCs in vitro. Researchers have made great progress in 10 ACS Paragon Plus Environment

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culturing MSCs in vitro. To realize the goal, conditions of microenvironment must be figured out and precisely controlled, which include physicochemical and biochemical parameters, as the Figure 3 shows.

2.3.1.2 Physicochemical

Physicochemical parameters consist of pH values, temperature, oxygen and carbon dioxide level, osmotic pressure and occasionally physical force such as hydrodynamic shear stress. pH value is typically kept at a strict 7.4, as a pH above or below 7.4 can cause harm to stem cells. Pluripotent stem cells are highly sensitive to the pH change in culture microenvironment, and the pH fluctuation can directly affect the growth, survival, proliferation and differentiation of cells. Temperature around 37℃ is most suitable for stem cells culture, as high temperature can do harm to stem cells and low temperature can influence the growth and metabolic rate. Osmotic pressure is a critical parameter which can cause effect on stem cell numbers and activity.25

Oxygen level is a considerable parameter in the sustainability of stem cells. To optimize the oxygen level, Dos Santos F. found that compared to 20% O2 (nonmaximal), 2% O2 (hypoxia) can better maintain BM MSC characteristic immunophenotype and differentiative potential, moreover, 2% O2 level can lead to a more efficient bone marrow MSCs expansion.26 Mohyeldin found the effects of hypoxia to varies stem cells, and other gas other than oxygen such as NO may be potential regulatory factors to stem cells growth.27



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Because of the lack of vessels in vitro culture, essential substance transport must rely on mixing the matrix sometimes. Thus, hydrodynamic shear stress on stem cells is an important issue in controlling the fate of stem cells. However, stem cells in vitro culture conditions cannot be precisely monitored and controlled by traditional culture methods.

2.3.1.3 Biochemical

Biochemical parameters generally consist of nutrients, metabolic waste and some special substances such as growth factors and Cytokines. To better understand connections between them, scientists have done a wide variety of research.

Fernandes T. G. optimized the culture conditions of embryonic stem cells and evaluated the cells biochemical factors in the growth microenvironment including cell planting density, cell nutrients and metabolic by-products lactic acid effects on the growth of stem cells. The results showed that the serum-free environment had no effect on cell metabolism, differentiation and adhesion, but was more beneficial to cell metabolism. In low seeding culture, the authors found that glutamine was the preferred energy source for embryonic stem cells expansion.28

The effects of pH and lactic acid levels on pluripotent stem cells have been studied in detail. Lactic acid itself had little effect on the differentiation of pluripotent stem cells, but too much lactic acid could inhibit cell activity and proliferation. The biggest damage lactic acid did to cells came from its accumulation causing the down regulation of the microenvironment pH value.29 12 ACS Paragon Plus Environment

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Nowadays, many defined growth factors such as bFGF are added into culture substrate to regulate stem cell growth.30 Growth factors and cytokines are signals that mainly regulate the fate of cells. Seeding density is another critical factor to ensure maximum yield. Sotiropoulou found that low density MSC maintain high proliferation rate.31 Normally the stem cells are anchorage-dependent cells that proliferate in a single layer. In the culture of porcine bone-marrow-derived primary mesenchymal stem cells, Cytodex-I microcarriers were used for stem cell adhesion and proliferation, making the way of the cultivation of stem cells from anchorage-dependent into cytokine-dependent, showing the successful transition from two-dimensional to three-dimensional cultivation model.32 Scientists have paid efforts to study and mimic the in vivo niches for the reason that stem cells proliferation and differentiation must be precisely controlled to satisfy the need of clinical use both in the number and the state.

3. Smart-bioreactor for stem cell proliferation and differentiation

3.1 The development process of bioreactor

Tissue engineering and regenerative medicine requires a large amount of cells for clinical treatment.33 However, the cells obtained from donors is often limited. Therefore, the cells need to be cultured and expand in vitro before its implantation into body. By now, proliferation and maintenance of adherent cell populations is usually performed in the form of monolayer cells in some containers, such as t-flasks, petri dishes, multi well plates. These disposable containers are easily used for aseptic 13 ACS Paragon Plus Environment


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operation with low cast. On the contrary, cultivation in such container provide limited increase in cell number. When large quantities are required, cells are often digested and passage into new container, while every passage require lots of labor and may decrease the cell viability. In addition, it’s almost impossible to monitor pH, pO2 and other environmental parameters of the medium in these container, and the effect of mass uneven distribution is also worth noting. Because of the drawback mention above, new culture system is urgently needed for cell cultivation in tissue engineering. Bioreactor, which provided an important way to solve these problems.

Bioreactor was firstly used in the fermentation of microorganism. The microorganism, such as saccharomyces and algae, are usually cultured suspending in bioreactor. In order to monitor the environment parameters in the bioreactor for further research to the growth characteristic of microorganism and get more product, several sensors were added to the bioreactor. For example, the pH, temperature and oxygen sensor, which enable real-time monitor to the cell cultivation process for longtime. With the development of tissue engineering, animal cells were cultivation in vitro and bioreactor was gradually used in animal cell cultivation. While the strategy of dynamic culture and computer-control bioreactor were used, which made it easier to exchange media and create a proper environment for animal cells, so that it could be possible for large scale cell culture in vitro. During the next four decades, more micro-structures were designed and studied to meet the need of different cells, which breeds a series of bioreactors.


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3.2 Bioreactor structure used in stem cells culture There have been many structures of bioreactor used in stem cells culture.33 And up to now, most of them adopt dynamic culture strategy to improve the mass transfer efficiency (gas, nutrient, waste). Figure 4 shows a classification of culture device structures used for animal cell., including mix vessels and perfusion vessels. The medium in mix vessels is mixed by mechanical force, such as stirring, rotating and waving, in order to realize a high transfer efficiency and more homogeneous cellular microenvironment. And in the perfusion system, cell is generally fixed while the medium is perfused and pass by. The perfusion system attempts to mimic blood flow and create a microenvironment like in vivo, making the cell growth density similar to the tissue density. In the following sections, we will introduce the characteristic of each bioreactor structure and review the application of these bioreactor to stem cell cultivation.

3.2.1 Mixed vessels

3.2.1.1 Spinner Flasks

Spinner flasks are generally glass vessels with side arms for medium and gas exchange, as the Figure 4(A) shows. Porous carriers with cells are fixed onto long needles and suspended in the medium. With the rotation of the magnetic stirrer, the media flow and mix, so that oxygen and nutrients evenly distributed. At present, the spinner flasks have been used in the human mesenchymal stem cells and adipose derived stem cells culture and to induce osteogenic differentiation.34-36 Comparison to 15 ACS Paragon Plus Environment


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static culture, spinner flasks can significantly promote cell growth and promote the generation of extracellular matrix and improve the uniformity of cells. The parameters of the spinner flasks are measured through the medium sample, while it’s difficult to be monitored in real time. And the limited scale of the cell number is also the shortcoming of the instrument.

3.2.1.2 Wave Bioreactors

The wave bioreactors usually use gas permeable bags with cells seeding in micro-carrier, as the Figure 4(B) shows. The culture vessel was placed on a shaker which provides a gentle motion and medium mixing. The disposable containing bag system is very convenient and hygienic in clinical use. With the properties of material used in the wave reactor further improved, a variety of stem cells have been successfully cultivated, such as human adipose derived stromal/stem cells, induced pluripotent stem cells, umbilical cord blood hematopoietic stem cells and human embryonic stem cells.37-40 In the study of ting and correia, the shapes of micro carriers are more uniform, and the induced agitation could well promote the differentiation of stem cells towards cardiomyocytes at faster kinetics and with higher yields compared with static culture and spinner. Similar as the spinner flasks, the parameters of the medium in wave bioreactor are difficult to monitor in real time, which hindered his larger application.

3.2.1.3 Rotating Wall Bioreactors


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The rotating wall bioreactor was initially designed to provide a micro-gravity environment similar to the space for cell culture, as the Figure 4(C) shows.41 It is mainly composed of a horizontally rotating cylinder filled with the culture substance. The cells were cultured in suspension micro-carriers or adhered to the walls. As the vessel rotates, the medium is sufficiently mixed, and a microgravity and micro shear force are produced to the cells, which thereby stimulating cell growth. At present, a variety of cells have been cultured in rotary wall bioreactors, such as mouse liver cells, mouse and human ESC, HSC, MSC osteogenic and chondrogenic differentiation. 42-45 Since the rotating-wall bioreactor can create a gas-liquid phase environment, it is used to induce differentiation of stem cells into lung cells and tracheal epithelial cells (TEC) and for clinical application.46-48 It is difficult for conventional rotating walls bioreactor to exchange medium, monitor micro environmental parameters and for large scale cultivation, however, the combination with perfusion system provides the possibility of more applications for rotating-wall bioreactors.

3.2.1.4 Stirred Tanks

The stirred tank bioreactor is generally a vessel equipped with a stirring impeller and is usually equipped with measuring and control units for maintaining the medium parameters such as temperature, pH, concentration of dissolved O2 and CO2 et al, as the Figure 4(D) shows. Cells are cultured suspension or seeded in micro-carriers in stirred tank. With the rotation of the impellers, the stirred bioreactor can easily mix the media evenly, creating a homogeneous and controllable microenvironment for 17 ACS Paragon Plus Environment


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cells growth. Because of its superior mass transport efficiency, stirred tank have been used in many cells to be cultured in vitro, such as human mesenchymal stem/stromal cells, human pluripotent stem cell, hematopoietic stem cells, human amniotic fluid stem cells et al.49-55 At the same time, large-scale stirred tank has also been used. Sullenbarger successfully cultured human MSC in the 50 L stirred bioreactor, and 25000 L reactor has also been used for antibody production.56-57 However, shear forces induced by agitators, turbulent flow and bubble break-up tend to cause damage to mammalian cells, which are mechanically sensitive, and related studies are reported in these articles. In order to reduce these damages in the stirred bioreactor, it should be specially designed so that the medium can be gently mixed without generating air bubbles. The usual method is to use larger diameter blades and antifoam agents.58 In the research of Tia Gareau, a certain range of shear force (

Bioreactor Synergy with 3D Scaffolds: New Era for Stem Cells Culture

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