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Biological and Medical Applications of Materials and Interfaces

Bacterial cellulose promotes long-term stemness of mESC Tina Tronser, Anna Laromaine, Anna Roig, and Pavel A. Levkin ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 20 Apr 2018 Downloaded from http://pubs.acs.org on April 20, 2018

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ACS Applied Materials & Interfaces

Bacterial cellulose promotes long-term stemness of mESC Tina Tronser1, Anna Laromaine2, Anna Roig2, Pavel A. Levkin1,3*

1

Karlsruhe Institute of Technology (KIT), Institute of Toxicology and Genetics (ITG), Hermann-von-Helmholtz-

Platz 1, 76344 Eggenstein-Leopoldshafen, Germany; 2

Institut de Ciència de Materials de Barcelona, Consejo Superior de Investigaciones Científicas (ICMAB-CSIC),

Campus de la UAB, 08193 Bellaterra, Catalunya, Spain 3

Karlsruhe Institute of Technology (KIT), Institute of Organic Chemistry, 76131 Karlsruhe, Germany

*Corresponding author: [email protected]

Keywords Bacterial cellulose, stem cells, cell differentiation, long-term maintenance, surface topography

Abstract Stem cells possess unique properties, such as the ability to self-renew and the potential to differentiate into an organism’s various cell types. These make them highly valuable in regenerative medicine and tissue engineering. Their properties are precisely regulated in vivo through complex mechanisms that include multiple cues arising from the cell interaction with the surrounding extracellular matrix, neighboring cells, and soluble factors. Although many research efforts have focused on developing systems and materials that mimic this complex microenvironment, the controlled regulation of differentiation and maintenance of stemness in vitro remains elusive. In this work we demonstrate for the first time that the nanofibrous bacterial cellulose (BC) membrane derived from Komagataeibacter Xylinus can inhibit the differentiation of mouse embryonic stem cells (mESC) under long-term conditions (17 days), improving their MEF-free cultivation in comparison to the MEF-supported conventional culture. The maintained cells´ pluripotency was confirmed by the mESCs’ ability to differentiate into the 3 germ layers (endo-, meso-, and ectoderm) after having been cultured on BC membrane for 6 days. In addition, the culturing of mESCs on flexible, free-standing BC membranes enables the quick and facile manipulation and transfer of stem cells between culture dishes, both of which significantly facilitate the use of stem cells in routine culture and various applications.

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To investigate the influence of the structural and topographical properties of the cellulose on stem cell differentiation, we used cellulose membranes differing in membrane thickness, porosity and surface roughness. This work identifies bacterial cellulose as a novel convenient and flexible membrane material enabling long-term maintenance of mESCs’ stemness and significantly facilitating the handling and culturing of stem cells. Introduction Stem cells´ unique properties such as their self-renewal ability and potential to differentiate into various cell types make them highly valuable in developmental, biomedical research and tissue engineering.1-2 In vivo, cell stemness is controlled via a complex system of multiple cues coming from the microenvironment such as interaction with neighboring cells, extracellular matrix, or soluble factors.3 The development of systems that could mimic such in vivo microenvironment to maintain stemness in vitro is highly essential, yet it remains elusive and challenging due to the great complexity of the cellular microenvironment and multitude of requirements important to maintaining stemness.4-7 In case of mouse embryonic stem cells (mESC), these requests are: 1) precoating the culture vessel with gelatin to provide attachment sites, 2) adding the cytokine leukemia inhibitory factor (LIF) inhibiting differentiation, 3) regular passaging (every 48h) to prevent overgrowth (known to induce differentiation) and, 4) the use of mitotically inactivated mouse embryonic fibroblasts (MEFs), known to increase cell attachment and secrete soluble factors maintaining stemness.8-10 Furthermore, the use of MEFs involves further purification steps in order to obtain a pure stem cell population.11-12 In addition, the inactivation of MEFs is primarily enabled through mitomycin C treatment or irradiation, and it can carry the risk of cytotoxic side effects on stem cells due to residual mitomycin C.13 All in all, this demonstrates how laborious, timeconsuming, and costly the culturing of mESCs is, and despite precise control of these requirements, maintaining stemness remains difficult. Few alternative artificial materials have been developed and applied for maintaining mouse embryonic stem cell (mESC) culture.6 Thus, there is still a great need for the development of artificial systems and materials mimicking the in vivo microenvironment and, therefore, capable of maintaining the undifferentiated state of stem cells. Various studies showed that stem cells can be strongly influenced in their differentiation behavior by attachment to materials exhibiting specific surface topography, porosity, roughness, and fibrillary structure.7, 14-15 Cellulose is a unique, naturally fibrillary and porous material that can be derived from various plants, fungi and bacteria. Thereby much progress has been made in the development of plantbased scaffold materials for biomedical applications serving as wound dressing material.16 Furthermore bacterial cellulose is playing an increasingly important role in research fields such as tissue engineering, clinical, and transplantation research.17-18 Bacterial cellulose (BC) possesses various advantages over 2 ACS Paragon Plus Environment

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cellulose from other origins. One advantage is its purity, further enhancing its biocompatibility.18-19 In addition, BC membranes are formed by an intertwined network of fibers of fine porosity composed of fibrils with diameters measuring tenths of nanometers, much thinner than plant cellulose.19-21 BC exhibits high crystallinity, mechanical strength, high water-holding capacity, broad chemical modifying ability, biodegradability, and the ability of 3D molding during synthesis.

18, 20-22

BC is most commonly

synthesized under static culture conditions, generating a cellulose membrane at the liquid-air interface. 1921

BC membrane thickness can be modulated by the cultivation time, and its porosity, roughness, and

mechanical properties through its drying method.

21

Thanks to BC’s advantages, it finds applications in

tissue engineering and as a skin substitute and wound-dressing material, exhibiting higher complement activation than conventional graft materials.

19-20, 23

Furthermore, previous studies showed morphological

similarities between BC and the extracellular matrix protein collagen, indicating comparable support in cell growth or even possible maintenance of the undifferentiated state of stem cells through the BC structure.24 In this work we exploit BC’s unique properties such as its purity, high biocompatibility, structural properties and morphological resemblance to collagen to culture mESC, enabling long-term maintenance of the cells’ undifferentiated state under MEF-free culture conditions. At the same time, the mESCs’ culture on the free-standing BC membrane enabled us to easily handle and manipulate stem cells while significantly facilitating routine culture. Results Fabricating bacterial cellulose membranes Bacterial cellulose (BC) is produced by culturing the bacterial strain Komagataeibacter Xylinus in a static culture resulting in the formation of a cellulose layer at the liquid-air interface. The membranes were cleaned and dried before further use by following different drying procedures, leading to different structural properties and mechanical characteristics studied extensively in a previous work.25 The subsequent experiments presented here, if not stated differently, were conducted at room-temperature dried and rewetted bacterial cellulose membrane (RT-BC wet). To investigate the BC membrane’s influence on the maintenance of stem cells’ stemness in an MEF-free culture system, we used a transgenic mouse embryonic stem cell line (mESC Oct4-eGFP) stably expressing eGFP (enhanced green fluorescent protein) fused to the pluripotency gene Oct4 (octamerbinding transcription factor 4). 26 This enables an immediate read-out of the Oct4 expression via the GFP level, which provides a straightforward indication of the stemness of the mESC Oct4-eGFP.



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The in vitro systems conventionally used for culturing mESC Oct4-eGFP (Figures 1A, B and Figure S1A) require gelatin coating of the culture surface to provide attachment sites, as well as the use of mitotically inactivated MEFs, known to further enhance cell attachment and secrete soluble factors maintaining stemness. Furthermore, the cytokine LIF - known to inhibit differentiation - must be added.

27

This

procedure is laborious, time-consuming and costly, and even when these requirements are carefully fulfilled, maintaining stemness remains difficult. Artificial materials with unknown properties can influence cell viability and result in increased cytotoxicity. Hence, to assess the biocompatibility of the respective artificial material, it is essential to assess the cell viability. Thus we measured the viability of the mESC Oct4-eGFP upon culturing under the aforementioned conventional conditions and on the BC-RT wet membranes (Figure 1C) by staining with propidium iodide (PI) to assess the number of dead cells, and Hoechst 33342 to assess total cell numbers. For better comparability between the conventional culture and culture on BC, the values obtained from dead (PI-positive, Hoechst-33342 positive) and viable cells (PI-negative, Hoechst-positive) were normalized to the total cell count of the respective samples. Upon culture on the BC-RT wet, the percentage of dead cells measured at 2h cultivation time was higher than that upon the conventional culture (Figure 1C). However the overall increase in the percentage of dead cells from 2h to 24h was lower for culture on BC with 6% (2h: 22%; 48h: 28%) than for conventional culture with 16% (2h: 4%; 24h: 20%). This could be an effect of increased mechanical stress of the cells during the initial cultivation time due to the BC’s unusual surface properties and lack of previous cell conditioning to the surface. Further experiments as for example immunostaining of the cytoskeleton need to be conducted to validate this hypothesis. We also assessed the growth rate from 2h to 48h by measuring the size of the mESC Oct4-eGFP colonies (Figure S1B), which revealed greater growth of the mESCs colony on BC than on the conventional culture. Hence, despite initial stress inducing cell death, the maintenance of viable cells and increased proliferation over a 24h culture were achieved on BC membranes together with a strong reduction in culture requirements compared to the conventional mESC Oct4-eGFP culture. In addition, cultivating mESCs on the free-standing BC membrane allowed facile cell transfer to a culture dish containing fresh medium (Video S1) further simplifying routine cell culturing by reducing the number of steps needed during medium exchange in standard cultures.


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Figure 1. (A) Schematic representation demonstrating artificial culture systems (schematics in gray boxes) mimicking great complexity of the in vivo microenvironment of stem cells (left schematic, blue box) in order to enable the maintenance of stemness. Conventional culture methods require gelatin coating, adding inactivated MEFs and LIF to promote stemness (upper schematic, upper gray box). Culturing mESC on bacterial cellulose possessing adjustable and variable structural properties (roughness, topography, porosity) (bottom schematic, bottom gray box) inhibits mESCs’ differentiation while resulting in significantly fewer requirements than conventional cultures (B) 3D reconstruction of confocal images showing conventional culture conditions (upper image) with inactivated MEFs (blue) and mESC Oct4-eGFP (green) as well as mESC Oct4-eGFP (green) cultured on BC-RT wet (red). (C) Viability of mESC Oct4-eGFP upon culture under conventional conditions (left graph) and culture on BC-RT wet (right graph). Assessment of dead cells via PI staining. N=3. Statistical significance: t-test, * indicates p-value ≤0.05.

Next, we wanted to investigate how the BC membrane’s microstructural properties influence mESC stemness. For that, we measured the roundness of mESC colonies, known to be a morphological indicator of stemness, with reduced roundness indicating differentiation (Figure S1C).28 Those results demonstrate a decrease in roundness of mESC upon culture under conventional conditions, whereas culture on BC 5 ACS Paragon Plus Environment


results in the roundness being maintained over a 48h time course. Furthermore, we used the expression of GFP as an indication for stemness due to its stable fusion to Oct4. To quantify the GFP expression, we measured the mean fluorescence intensity and counted the number of completely undifferentiated colonies (GFP+), mixed colonies (GFP+ and GFP-), and fully differentiated colonies (GFP-) (Figures 2A, 2B). Figure 2A shows an increase in the mean fluorescence intensity of mESC Oct4-eGFP cultured on BC from 1 at 2h to 1.5 at 24h, whereas under conventional culture conditions the mean fluorescence intensity weakened from 0.7 at 2h to 0.6 at 24h. This demonstrates better maintenance of stemness and reduced spontaneous differentiation, most likely due to the cellulose membrane’s specific surface properties. The numbers of GFP +, mixed, and GFP- colonies we counted support this observation, showing a reduction in GFP+ colonies by only ~8% upon culture on the BC and in contrast to under conventional culture conditions by ~20% during 72h cultivation time. These findings indicate a reduction in spontaneous differentiation upon short time culture through the BC. The long-term maintenance of mESCs’ undifferentiated state remains challenging unless the culture requirements of standard in vitro systems are precisely regulated. We thus aimed to investigate the effect of the BC membranes on the differentiation and maintenance of stemness of mESC Oct4-eGFP under long-term culture conditions (17 days). The mESC Oct4-eGFP were cultured under conventional culture conditions and on BC, both in 5 mL medium supplemented with LIF, without further passaging for 17 days. To ensure a sufficient supply of nutrients during the experiment, medium was changed daily and LIF added freshly each time. We counted the number of colonies per mm2 in both samples and observed a strong increase in mixed and GFP- colonies when mESC Oct4-eGFP were cultured under conventional conditions and a decrease in GFP+ colonies from 99% at day 0 to 8% at day 17. In contrast to that, culture on the BC significantly delayed the mESCs’ spontaneous differentiation even under long-term culture conditions, with a reduction in GFP+ colonies from 99% at day 0 to only 48% at day 17 (Figure 2C). To prove the observed effect of BC on maintaining stemness and the potential of the mESC Oct4-eGFP to differentiate into cell derivatives of all three germ layers, we performed immunofluorescence staining for markers of the germ layers: endo-, meso- and ectoderm (Figures 2D, S1E). For this, mESC Oct4-eGFP were cultured under conventional conditions and on the BC membrane for 6 days before being detached and pipetted onto the inner side of the lid of a Petri dish. The Petri dish lid was immediately inverted to allow the culture of mESCs in hanging drops and, hence, the formation of mESC aggregates called embryoid bodies (EBs) that recapitulate early embryonic development by differentiating into derivatives of all germ layers. After 48h culture in hanging droplets, the EBs were collected and transferred to fibronectin-coated cover slips for a further 12-day culture resulting in attachment, outgrowth, and differentiation of the EBs into cell derivatives of the respective germ layers. Following the EBs’ outgrowth and differentiation, immunofluorescence staining was done for FoxA2, Brachyury, β-III6 ACS Paragon Plus Environment

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Tubulin - markers for endo-, meso- and ectoderm, respectively. Representative images of immunofluorescence staining for each germ layer’s marker are illustrated in Figure 2D, proving maintenance of the stemness of the respective mESCs after being cultured for 6 days on the BC.

Figure 2. Assessment of stemness of mESC Oct4-eGFP upon culture on BC-RT wet in comparison to conventional culture conditions. (A) Measurement of mean fluorescence intensity of mESC Oct4-eGFP under conventional culture conditions and under culture on BC-RT wet. Statistical significance: t-test, * indicates p-value ≤0.05. (B) Percentage of GFP + , mixed and GFP – colonies per mm2 at 2h, 48h and 72h under conventional culture and on BC-RT wet. Right side: representative images of GFP+, mixed and GFP- colonies. (C) Percentage of GFP+, mixed and GFPcolonies under long term culture conditions (for 17 days). N=3, n > 50. (D) Immunofluorescence staining showing potential of mESC Oct4-eGFP to differentiate into 3 germ layers (endo-, meso-, and ectoderm) after being cultured on BC-RT wet. Cells were culture on BC-RT wet for 6 days, detached and cultured for 48h using hanging drop method to form embryoid bodies (EBs). EBs were transferred to fibronectin-coated cover slips and cultured for 12



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days. Subsequent immunofluorescence staining of endo-, meso- and ectoderm markers (Fox A2, Brachyury and βIII-Tubulin respectively) and cell nucleus (DAPI) was performed. Scale bar 100 µm.

Next, we wanted to elucidate the underlying mechanism leading to better maintenance and inhibition of differentiation upon culture on BC. Unlike plant-derived cellulose, BC exhibits higher purity. We assumed this difference could be responsible for the influence on stemness maintenance we had observed. To investigate that hypothesis, we compared mESC Oct4-eGFP cultured under conventional conditions on BC-RT wet and on commercially available, plant-derived cellulose (filter paper (FP); 180 µm thickness, surface roughness (Sa) 4.93 µm, pore size 11 µm, fiber diameter 12 µm ± 2.6). Differences in the mean fluorescence intensity of mESC Oct4-eGFP colonies appeared between mESC grown on BC-RT wet and on FP (Figure 3A). The BC-RT wet and purchased FP differ both in their origin and purity and their microstructural properties. In particular, BC is known as a form of nanocellulose since their cellulose fibrils possess a fiber diameter in the nanoscale (20-50 nm) in contrast to the micron scale diameter (10-50 µm) of plant cellulose. Based on this, the differences between FP and BC-RT wet noted are also likely to arise from differences in thickness and topographical structure than solely from the cellulose’s origin (Figure 3B). To investigate the influence of the microstructural and mechanical properties of the cellulose on stem cell differentiation, we used various BC membranes that were dried via different drying procedures, which in turn changed the BC membrane thickness, porosity, and surface roughness. We employed wet, never-dried BC (BC-W), freeze-dried BC (BC-FD), and room temperature-dried BC (BC-RT), whereas the latter was either used rewetted (BC-RT wet) or in a dry state (BC-RT dry). The membranes’ thickness and surface roughness were measured via confocal microscopy and optical profilometry. Figure 3B clearly shows the effect the specific means of drying (BC-W, BC-FD, BC-RT dry) and rewetting (BC-RT wet) has on the thickness, ranging from 200 µm to 16 µm 25, and on surface roughness (Sa), ranging from 7.65 µm to 0.59 µm. First, we examined the cell attachment to the different cellulose derivatives after 24h. mESC were therefore seeded in the same cell concentration on the various BC membranes, imaged via fluorescence microscopy, and evaluated by counting the number of attached cells and normalizing those values to the initial cell concentration. The best cell attachment (32%) was achieved on room temperature-dried BC independent of rewetting (Figure 3C; BC-RT wet and BC-RT dry; both 32%) and the lowest cell attachment (7%) was achieved on the freeze-dried BC (BC-FD), indicating that surface roughness is an important factor in cell attachment, as the thickness of BC-RT wet differs only slightly from that of BCFD. Next, we checked the different cellulose derivatives for their ability to promote and maintain stemness, by measuring the mean fluorescence intensity of mESC cultured on the respective cellulose samples for 48h. To maintain an equal and sufficient nutrient supply between the respective BC membranes, 5 mL medium with LIF was added to all samples after seeding. The highest mean 8 ACS Paragon Plus Environment

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fluorescence intensity was measured on BC-RT wet, whereas its completely dry state (BC-RT dry) revealed reduced intensity. Also mESC grown on dry BC-FD also showed reduced mean fluorescence intensity that is comparable to the BC-RT dry values (Figure 3D). One cause for these differences could result from the varying capacities of the wetted and dry BC-RT membranes to absorb liquids, with the BC-RT membrane revealing greater absorption capacity (37.3) in the dry state than after rewetting of the cellulose membrane.25 This in turn could result in a faster rate of cell-suspension absorption accompanied by potentially stronger forces acting on the mESC during absorption by the BC membrane, inducing differentiation. Differences in the BC membranes’ surface roughness could also result, through enhanced cell attachment and hence cytoskeleton organization, in the observed differences in maintaining stemness (BC-RT wet: 1.29 µm; BC-FD: 7.66 µm).

14, 29-30

However, further experiments showing cell attachment

over time have to be performed to elucidate the underlying mechanism resulting in the observed differences. Further, we tested the BC’s ability to act as storage for nutrients and soluble factors. This was done by preincubating the BC-RT wet membranes with culture medium obtained from 24h mESC culture (BC-RT wet: Medium) or with LIF alone (BC-RT wet: LIF) for 5 min prior cell seeding (Figure S2A). Surprisingly, both preconditioned samples (BC-RT wet: Medium; BC-RT wet: LIF) exhibited lower mean fluorescence intensity than the non-preconditioned sample (BC-RT wet), suggesting increased absorption of the metabolites or soluble factors inducing differentiation.



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Figure 3. Assessment of stemness of mESC Oct4-eGFP upon culture on various cellulose samples. (A) Mean fluorescence intensity of mESC Oct4-eGFP cultured on BC-RT wet in comparison to culture on filter paper: Whatman® qualitative filter paper Grade 1. Cultivation time was 2h – 48h. (B) Comparison of thickness and surface roughness between filter paper and bacterial cellulose samples dried via various methods. BC-W: never-dried bacterial cellulose. BC-RT: Room temperature-dried bacterial cellulose. Deployed for experiments in dry state (BCRT dry) and wetted state (BC-RT wet; wetted with di-water). BC-FD: freeze-dried bacterial cellulose. (C) Cell attachment at 24h of culture on different bacterial cellulose samples dried via various methods. (D) Mean fluorescence intensity of mESC Oct4-eGFP upon culture on differently-dried bacterial cellulose samples. Cultivation times were 2h – 48h. N=3. Statistical significance: t-test, * indicates p-value ≤0.05.

Our previous results demonstrate the strongest effect on mESC stemness resulting from culture on BC-RT wet membranes, indicating influence through the surface properties and sorption capacity of the cell suspension through the material. To demonstrate the influence of surface properties on stemness, we masked the surface structure by applying a gelatin coating on the BC-RT wet membrane prior cell seeding (Figure S2B). The gelatin masking resulted in significantly less mean fluorescence intensity than uncoated BC-RT wet, supporting our earlier results regarding the influence of surface topography (Figures 3B, D). Previous studies showed an effect of different surface structures, such as a porous fibrillary structure, on cell attachment through infiltration into porous scaffold and degradation of the surrounding matrix by the cells, resulting in controlled differentiation.31-32 Via confocal microscopy, however, no mESC infiltration into the BC-RT wet membranes was detectable, as we anticipated due to the lower porosity of the surface 10 ACS Paragon Plus Environment

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layer of membranes dried at room temperature (Figure S2D). To investigate further possible infiltration and degradation of the cellulose by the mESC, we measured the thickness of the cellulose membranes before (BC-RT wet: mESC) and after (BC-RT wet: mESC +Trypsin) mESC cell detachment (Figure S2C). As controls, we used an empty BC membrane incubated under the same conditions (BC-RT wet: control; no cells; 5 mL + LIF; 48 h) having a thickness of ~31 µm. After detaching the mESC Oct4-eGFP using trypsin, we observed partial dissolution of the BC membrane, indicated by its thickness reduced to 22 µm (Figure S2C; BC-RT wet mESC +Trypsin). We can rule out the BC structure’s dissolution through enzymatic cleavage, since the treating of BC-RT wet with trypsin alone had revealed no significant loss of thickness (Figure S2C; BC-RT wet +Trypsin). The partial dissolution of the BC membrane we detected could result from loosening of the cellulose structure when the cells become detached due to a cell attachment via partial ingrowth of mESC Oct4-eGFP into the cellulose material, causing the cellulose fibers to partially detach upon trypsinization and with that, the cellulose structure to become thinner. Another possible cause for the dissolution of the BC membrane could be due to proteins secreted by the mESC leading BC membrane to partially dissociate allowing further ingrowth of the mESC. 31 To confirm these further experiments have to be conducted.

Discussion and Conclusion Bacterial cellulose is widely applied in tissue engineering, transplantation, and regenerative medicine as scaffold material used for cell transfer or the directed differentiation of cells based on its structural properties. The latter is especially important in the field of stem cell research, as the controlled differentiation and maintenance of stemness in vitro remains challenging, and many exacting requirements must be fulfilled to maintain the undifferentiated state in vitro. In case of the mouse embryonic stem cell culture, these requirements include precoating the culture vessel to provide sufficient cell attachment, medium supplementation with LIF (known to inhibit differentiation), regular cell passaging to prevent overgrowth, and the use of mitotically inactivated MEFs that provide further cell attachment sites and secrete soluble factors promoting stemness. All these steps make conventional mESC culture extremely laborious, time-consuming, and costly. Hence, the ability of nanocellulose of microbial origin to influence the maintenance of stemness and stem cell differentiation through its surface topography and microstructure makes it a promising material with the potential to revolutionize conventional stem cell cultures. In addition, mESC culture on a free-standing bacterial cellulose membrane not requiring the use of MEFs means the culturing requirements are significant reduced, and greatly facilitates handling and cell transfer in routine cultures. In this work, we exploit this potential by using bacterial cellulose membranes derived from Komagataeibacter Xylinus for mouse embryonic stem cell culture. We discovered the stemness-promoting effect of bacterial cellulose under short-term and long-term conditions. We 11 ACS Paragon Plus Environment


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successfully cultured mouse embryonic stem cells without requiring gelatin coating or mitotically inactivated MEFs for up to 72h, thus making the culture conditions much easier than those associated with conventional cultures. Moreover, the free-standing bacterial cellulose membrane simplified the routine cell culture by enabling the rapid transfer of mESCs between culture dishes (Video S1). Hence the BC membrane greatly simplifies the transfer and facitates the handling of mESCs during applications such as medium exchange in routine cultures and cell fixation. The ability to maintain stemness over short-term and long-term conditions may depend on the morphological similarities between bacterial cellulose and the collagen present in the extracellular matrix. 24

Moreover, the bacterial cellulose can act, as does the collagen, as a reservoir for growth factors (eg,

basic fibroblast growth factor (bFGF)) or soluble molecules affecting stemness).

33

To investigate this

hypothesis we measured the mean fluorescence intensity upon culture on non-conditioned BC membranes (BC-RT wet) and on LIF (BC-RT wet: LIF) and 24h culture medium (BC-RT wet: Medium) preconditioned BC membranes, respectively (Figure S2A). We observed reduced mean fluorescence intensity in both preconditioned BC membranes, indicating potentially increased absorption and storage of metabolites or of the soluble factors that impair stemness and induce differentiation (Figure S2A). Differences in cell attachment could be possible that in turn can lead to changes in the cytoskeleton of mESC, known to be an important regulator of cell differentiation by activating subsequent signaling pathways and transcription factors.

14, 29-30

However, to sustain this hypothesis further research

investigating the cell attachment needs to be conducted. We hypothesize that the BC membranes’ biochemical and physical properties such as its purity (lack of hemicellulose, lignin, or pectin), its nanofibrillary structure, and its porosity are main contributors to the maintenance of stemness upon culture on BC. Our results in Figure 3A reveal a BC-specific effect on stemness most likely brought about through its biochemical structure and greater purity compared to cellulose originating from plants (filter paper, 180 µm thickness, surface roughness (Sa) 4.93 µm) which showed less maintenance of stemness. However, we cannot claim that BC’s biochemical structure was the sole contributor from our findings, as BC and plant-derived cellulose differ significantly in their surface roughness and thickness as well (Figure 3B). Furthermore recent studies demonstrated the ability of plantderived nanofibrillary cellulose to act as scaffold material enabling culture of pluripotent stem cells and significantly maintaining the cells’ stemness. 34-35 As previous studies have shown, mESC differentiation and stemness can be controlled via surface structure, roughness, and porosity factors that rely on enhanced cell attachment and actin cytoskeleton reorganization - further suggesting that physical properties make an additional contribution to stemness. 14, 29-30

To investigate this hypothesis further, we employed BC membranes exhibiting the same biochemical 12 ACS Paragon Plus Environment

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properties due to sharing the same origin, but possessing different physical properties (surface roughness and thickness) depending on the drying method applied. The BC membranes employed revealed various thicknesses (16 µm, 31 µm, 40 µm, 200 µm) and surface roughness (0.59 µm, 1.29 µm, 7.66 µm, n.a.) on room temperature-dried BC (BC-RT dry or BC-RT wet), freeze-dried BC (BC-FD) and never-dried BC (BC-wet), respectively. We thus noted that mESC Oct4-eGFP cell attachment was highest on room temperature-dried BC, regardless of how it had been conditioned (dry state or wet state) before the experiment. We observed the lowest percentage of attached cells however upon seeding on freeze-dried BC (Figure 3C), which exhibited greater surface roughness than room temperature-dried BC (Figure 3B). 25

Such a rougher surface could trigger increased repulsion of mESC during initial seeding on BC-FD.

Regarding the maintenance of stemness, our results in Figure 3D indicate that, the physical properties of the BC membranes (like topography and roughness) also influence mESC development and differentiation. The varying drying routes applied here further affect, besides topography and roughness, the water absorbing capacity, elastic modulus and crystallinity of the BC membranes.25 Especially the water absorbing and swelling capacity of the BC membranes can have vital influence on nutrient and protein absorption as well as on cell attachment du to enhanced surface area, stiffness and roughness. We observed the strongest effect on promoting stemness on BC-RT membranes in conjunction with a significant difference between dry and prewetted BC-RT membranes, with the latter demonstrating greater maintenance. Prewetting the BC membranes causes the BC fibers to swell, thus expanding the surface area available for cells to attach and potentially preventing spontaneous differentiation. There is ample evidence of the effect of surface stiffness on differentiation and maintenance of stemness.14, 36-37 Further confirmation of those results is the surface structure’s masking effect via gelatin coating (Figure S2B), leading to lower mean fluorescence intensity and thus less maintenance of stemness. However, neverdried BC (BC-W) - with its greater porosity and surface area thanks to swollen BC fibers - exhibits reduced mESC stemness maintenance than BC-RT wet. This is attributable to reduced cell attachment (Figure 3C), which most likely results from the fibers having become saturated with water and hence less able to absorb a cell suspension during seeding, thus causing the mESCs’ displacement. We assume that the prewetted BC-RT exhibits a certain degree of surface roughness and swelling to permit good cell anchorage into the fibrous network, thus promoting stemness. Our Figure S2C results further illustrate the mESCs’ strong anchorage through partial ingrowth into the fibrous network, evident in the BC membrane’s slight dissolution after mESC detachment. However, to definitively identify the mechanism underlying enhanced cell attachment and maintenance of stemness, more research is needed. 38 In conclusion: our work demonstrates the ability of bacterial cellulose to promote the stemness of mouse embryonic stem cells over short-term and long-term conditions via its structural and mechanical properties that result in enhanced cell attachment and, hence, controlled differentiation. Our use of bacterial cellulose 13 ACS Paragon Plus Environment


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in a stem cell culture significantly minimized the necessary steps that usually need to be taken such as gelatin coating and the use of mitotically-inactivated MEFs of conventional, MEF-supported mouse embryonic stem cell cultures. Furthermore, the free-standing bacterial cellulose membrane enables fast transfer and the facile manipulation of mouse embryonic stem cells in routine cultures. Application of a bacterial cellulose membrane promotes the long-term maintenance of stemness while making mouse embryonic stem cell cultures much easier and less expensive to carry out than conventional methods. Future combinations of photopatterning methodologies or preparations of bacterial cellulose composites could make the investigation of stem cells upon different geometries and composite materials possible while maintaining the undifferentiated state of mouse embryonic stem cells through its surface topography. We believe that applying bacterial cellulose as a flexible, porous membrane material of high purity, biocompatibility, and enabling investigators to scale manufacturing offers the potential to advance stem cell culture and research. Material and Methods Production of Bacterial Cellulose: Bacterial strain Komagataeibacter Xylinus (KX) (ATCC 11142) was purchased from CECT (Spain). Glucose, peptone, yeast extract and agar were purchased from Conda Lab, and the Sodium hydroxide (NaOH), Sodium dihydrogen phosphate dodecahydrate (Na2HPO4•12H2O) and citric acid monohydrate were bought from Sigma Aldrich and used as received. KX were cultured in Hestrin-Schramm liquid medium (HS) with (w/v) 2% D-glucose, 0.5% peptone, 0.5% yeast extract, 0.115% citric acid, 0.68% Na2HPO4•12H2O. KX was first grown on a solid agar (1.5%) (Conda Lab) that was prepared in 10 cm diameter Petri dishes. Then a single colony was expanded in 5 mL HS liquid media at 30°C for 7 days in the dark. After the incubation time; a cellulose film could be observed on the top of the culture and the culture beneath was used as inoculum. The inoculum was transferred to an Erlenmeyer with 200 mL of liquid media. A thin membrane of bacterial cellulose (BC) grew on top of the liquid media over 5 days which was used for the subsequent experiments. Cleaning process: BC membranes harvested were immersed in ethanol. Subsequently they were transferred to deionized (DI) water and boiled for 40 min, then incubated with 0.1 M NaOH at 90°C for 20 min which was repeated two times before the membranes were finally neutralized with DI water for 24 h. Bacterial cellulose was dried via different drying procedures to make BC membranes possessing various characteristics. The following samples were prepared and labelled in relation to their drying methods. BCW: never dried bacterial cellulose was stored after sterilization in DI water. Room temperature drying (RT) bacterial cellulose membranes were placed into chromatography paper between two glass slides and then dried at room temperature for 4 days; obtaining BC-RT. For freeze-drying (FD); we plunge-freezed the samples in the accordion setup with liquid nitrogen for 5 min in a 50 mL falcon tube. Then, they were placed in the freeze-drier for 12 h and they were dried through sublimation of the solid water. The samples were freeze-dried in a LYOQUEST-85 freeze drier (Telstar) at -80 º C, below 0.005 mbar for 12 h; obtaining BC-FD. 14 ACS Paragon Plus Environment

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Characterization of BC membranes: The thickness of the films was measured with a digital micrometer (Mitutoyo QuantuMike IP65) as well as using confocal microscopy. The average sample thickness was determined by measuring at 10 different sites for each film. For the measurement of the surface roughness an optical profilometry (Zygo New View, Zygo/Ametek Germany) was used. mESC Oct4-eGFP culture: Mouse embryonic stem cells (mESC Oct4-eGFP) and mouse embryonic fibroblasts (MEFs) were kindly provided by Prof. Dr. Martin Bastmeyer (Karlsruhe Institute of Technology, Karlsruhe, Germany). mESC Oct4-eGFPs were cultured in DMEM (Gibco Life Technologies GmbH, Darmstadt, Germany) supplemented with 15% PANSera ES (PAN-Biotech GmbH, Aidenbach, Germany). 1% Penicillin/Streptomycin (Gibco Life Technologies GmbH, Darmstadt, Germany), 1x NEAA (100x Stock, Gibco Life Technologies GmbH, Darmstadt, Germany) and 0.1 mM β-mercaptoethanol (Alfa Aesar, Karlsruhe, Germany). The culture flasks were previous to mESC culture precoated with 0.1% porcine gelatin in PBS and mitotically-inactivated MEFs were seeded. For mitotical inactivation, MEFs were incubated in 10µg/mL Mitomycin C (Santa Cruz Biotechnology, Inc., Dallas, USA) for 2-3h. The mESC were passaged every second day and per 5 mL culture medium 150µL of leukemia inhibitory factor (LIF, sterile filtered supernatant of HEK293 stably transfected with a LIF expression plasmid, provided by Prof. Dr. Martin Bastmeyer, Karlsruhe Institute of Technology, Karlsruhe, Germany) was added. Preplating of mESC Oct4-eGFP: To isolate mESC Oct4-eGFP from the co-cultured MEFs, the cells were preplated before each individual experiment. The cell suspension containing mESC Oct4-eGFP and MEFs was transferred to a non-coated Petri dish and incubated for 30 min at 37°C and 5% CO2. Due to the slower cell adherence of mESCs to non-coated surface than MEFs, mESC Oct4-eGFP can be collected from the supernatant while the MEFs remain attached to the surface. Seeding and culture of mESC Oct4-eGFP on bacterial cellulose/filterpaper: mESCs were seeded at the required concentration in a volume of 500 µL on the bacterial cellulose membranes or on the filterpaper (Whatman® qualitative filter paper Grade 1) in a 60 mm Petri dish and allowed to set for 90 sec. To reduce the displacement of cell suspension during the seeding procedure on wet bacterial cellulose samples (BC-W and BC-RT wet), excess water was aspirated from the wet BC samples beforehand. 4.5 mL of the stem cell culture medium and 150 µL of LIF were added covering the bacterial cellulose membrane completely. The medium was changed every second day by transferring the bacterial cellulose membrane with mESC to a fresh 60 mm Petri dish containing 5 mL culture medium and 150 µL LIF. For control purposes, mESCs were cultured under conventional conditions as mentioned above. Viability staining: The mESC were seeded on the BC membranes as mentioned above and cultured for the respective days in the presence of Hoechst 33342 in a dilution of 1:10.000 (1µg/mL; Molecular Probes, Thermo Fisher Scientific Inc., Massachusetts, USA) to stain the cell nucleus and in the presence of Propidium iodide (PI) at a concentration of 100 nM (Invitrogen, California, USA) to stain dead cells. Images were taken at the respective time points and cells were counted to assess viability. A further experiment (data not shown) 15 ACS Paragon Plus Environment


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assessing the stains’ toxicity on mESCs revealed no difference in mESC viability between cultured with and without stains (staining only at respective time points). Embryoid body formation: The mESC were cultured on BC-RT wet for 6 days with regular medium changes. mESCs cultured under conventional culture conditions served as controls. The mESCs were detached from the samples using trypsin and to form embryoid bodies (EBs) cultured for 48h via the hanging drop method by pipetting 25 µL drops containing cells (0.16 x 106 cells/mL) onto the Petri dish lid, then immediately turning and placing the lid on a PBS-filled Petri dish. The formed EBs were collected from the Petri dish lid and transferred onto a fibronectin (10µg/mL)-coated coverslip, enabling outgrowth and spontaneous differentiation into all 3 germ layers (endo-, meso-, ectoderm) for 12 days in conjunction with regular medium changes. Following outgrowth immunofluorescence staining for markers of endo-, meso- and ectoderm (Fox A2, Brachyury, β-III Tubulin) was performed. Immunofluorescence staining: The outgrown EBs were fixed on coverslips using 3.7% paraformaldehyde in PBS for 15 min, and permeabilized using 0.1% Triton-X 100 in PBS for 15 min. The respective EB samples were incubated for 1h with the following primary antibodies, each in a 1:500 dilution in 1% BSA/PBS: goat anti-Fox A2 (HNF 3β) (R&D systems, Minneapolis, USA); goat anti-brachyury (R&D systems, Minneapolis, USA); rabbit anti-β-III-Tubulin (TUj1) (Sigma-Aldrich, Munich, Germany). The samples were washed with PBS, and incubated for 1h in the dark with the respective secondary antibodies in 1% BSA/PBS, each in 1:200 dilution: donkey anti-goat Cy3 and goat anti-rabbit Cy3 (Jackson Immunoresearch, West Grove, USA). DAPI (Molecular Probes, Thermo Fisher Scientific Inc., Massachusetts, USA) in 1:10000 dilution was added to all samples along with the secondary antibodies. Image acquisition and Analysis: Fluorescence images were taken using the microscope Keyence BZ9000 (Keyence, Osaka, Japan) and confocal microscope images were taken using Leica SPE confocal microscope (Leica Microsystems CMS GmbH, Mannheim, Germany). The exposure times were set and kept the same for all conditions throughout all experiments and repetitions. To assess mESC stemness, the intrinsic Oct4-eGFP signal was used and evaluated as follows: for the percentage of pluripotent mESC, GFP-positive (complete colony fluorescent), mixed (colony showing fluorescent and non-fluorescent regions), and GFP-negative colonies (complete non-fluorescent colony) were counted. For quantifying reasons we measured mean fluorescence intensity using ImageJ (https://imagej.nih.gov/ij/). The colony size and roundness of the mESC colonies were assessed using ImageJ. Statistical analysis: All quantitative data was normalized and depicted as mean ± SD. At least 3 individual repetitions were performed for each experimental set-up and used for statistical analysis. All data sets were statistically analyzed using two-tailed student’s t-test using OriginPro (OriginLab Corperation). P-values < 0.05 were considered statistically significant. Acknowledgements 16 ACS Paragon Plus Environment

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The research was supported by the Helmholtz Association’s Initiative and Networking Fund (Grant No. HGF-ERC-0016) and the Spanish Ministry of Economy, Industry and Competitiveness through the MAT2015-64442-R project and the Severo Ochoa Programme for Centers of Excellence in R&D (SEV2015-0496) and the Generalitat de Catalunya 2017SGR765. The authors are grateful to Prof. Dr. Martin Bastmeyer, Karlsruhe Institute of Technology (KIT), Karlsruhe Germany for providing the mouse embryonic stem cell line (mESC Oct4-eGFP) as well as the mouse embryonic fibroblast cell line (MEFs). References 1. Kobel, S.; Lutolf, M. P., High-throughput methods to define complex stem cell niches. Biotechniques 2010, 48 (4). 2. Laustriat, D.; Gide, J.; Peschanski, M., Human pluripotent stem cells in drug discovery and predictive toxicology. Biochem Soc T 2010, 38, 1051-1057. 3. Watt, F. M.; Hogan, B. L., Out of Eden: stem cells and their niches. Science 2000, 287 (5457), 1427-30. 4. Balikov, D. A.; Crowder, S. W.; Boire, T. C.; Lee, J. B.; Gupta, M. K.; Fenix, A. M.; Lewis, H. N.; Ambrose, C. M.; Short, P. A.; Kim, C. S.; Burnette, D. T.; Reilly, M. A.; Murthy, N. S.; Kang, M. L.; Kim, W. S.; Sung, H. J., Tunable Surface Repellency Maintains Stemness and Redox Capacity of Human Mesenchymal Stem Cells. ACS Appl Mater Interfaces 2017, 9 (27), 22994-23006. 5. Alberti, K.; Davey, R. E.; Onishi, K.; George, S.; Salchert, K.; Seib, F. P.; Bornhauser, M.; Pompe, T.; Nagy, A.; Werner, C.; Zandstra, P. W., Functional immobilization of signaling proteins enables control of stem cell fate. Nat Methods 2008, 5 (7), 645-650. 6. Joddar, B.; Ito, Y., Artificial niche substrates for embryonic and induced pluripotent stem cell cultures. J Biotechnol 2013, 168 (2), 218-228. 7. Moraes, C.; Chen, J. H.; Sun, Y.; Simmons, C. A., Microfabricated arrays for high-throughput screening of cellular response to cyclic substrate deformation. Lab Chip 2010, 10 (2), 227-234. 8. Efe, J. A.; Ding, S., The evolving biology of small molecules: controlling cell fate and identity. Philos T R Soc B 2011, 366 (1575), 2208-2221. 9. Llames, S.; Garcia-Perez, E.; Meana, A.; Larcher, F.; del Rio, M., Feeder Layer Cell Actions and Applications. Tissue Eng Part B-Re 2015, 21 (4), 345-353. 10. van der Sanden, B.; Dhobb, M.; Berger, F.; Wion, D., Optimizing stem cell culture. J Cell Biochem 2010, 111 (4), 801-7. 11. Higuchi, A.; Ling, Q. D.; Kumar, S.; Munusamy, M.; Alarfajj, A. A.; Umezawa, A.; Wu, G. J., Design of polymeric materials for culturing human pluripotent stem cells: Progress toward feeder-free and xeno-free culturing. Prog Polym Sci 2014, 39 (7), 1348-1374. 12. Rodin, S.; Antonsson, L.; Hovatta, O.; Tryggvason, K., Monolayer culturing and cloning of human pluripotent stem cells on laminin-521-based matrices under xeno-free and chemically defined conditions. Nat Protoc 2014, 9 (10), 2354-68. 13. Acquarone, M.; de Melo, T. M.; Meireles, F.; Brito-Moreira, J.; Oliveira, G.; Ferreira, S. T.; Castro, N. G.; Tovar-Moll, F.; Houzel, J. C.; Rehen, S. K., Mitomycin-treated undifferentiated embryonic 17 ACS Paragon Plus Environment


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