Cell Culture Scaffolds Research Articles

A practical guide to microfabrication and patterning of hydrogels for biomimetic cell culture scaffolds

This review article describes microfabrication techniques to define chemical, mechanical and structural patterns in hydrogels and how these can be used to prepare in vivo like, i.e. biomimetic, cell culture scaffolds. Hydrogels are attractive materials for 3D cell cultures as they provide ideal culture conditions and they are becoming more prominently used. Single material gels without any modifications do however have their limitation in use and much can be gained by in improving the in vivo resemblance of simple hydrogel cell culture scaffolds. This review article discusses the most commonly used cross-linking strategies used for hydrogel-based culture scaffolds and gives a brief introduction to microfabrication methods that can be used to define chemical, mechanical and structural patterns in hydrogels with micrometre resolution. The review article also describes a selection of literature references using these microfabrication techniques to prepare organ and disease models with controlled cell adhesion, proliferation and migration. It is intended to serve as an introduction to microfabrication of hydrogels and an inspiration for novel interdisciplinary research projects.

Rapid and Detergent-Free Decellularization of Cartilage.

The use of decellularized tissues or organs as cell culture scaffolds has proven to be a promising approach for tissue engineering and regenerative medicine, as these decellularized tissues can provide the instructive niche for cell differentiation and functions. Cartilage is a largely avascular tissue with limited regenerative capacity. Lesions caused by arthritis can lead to severe cartilage degeneration. Previous studies have indicated that decellularized cartilage can be used as scaffolds that support the chondrogenic differentiation of adult stem cells. However, these decellularization protocols all require the use of denaturing agents, such as high salt and detergents, that lead to the artifactual disruption of the chemical and physical integrity of the tissue microenvironment. Here, we established a new decellularization method for cartilage, through a combined effect of freezing-thawing, sectioning, and sonication in water. This protocol achieved the complete removal of cells within minutes, instead of hours or days required by existing procedures, and does not use any detergent. The resulting decellularized cartilage preserved the native ultrastructure and biochemical contents, including glycosaminoglycans, which is typically depleted by traditional decellularization methods. Human mesenchymal stem cells could readily adhere onto the decellularized cartilage. Together, this work unveils a simple new method for decellularizing cartilage, which will be useful in studying how tissue microenvironment supports chondrocyte growth and functions. Impact statement In this study, we develop a simple, fast cartilage decellularization method that does not require any detergent, so that the decellularized cartilage chemistry is preserved. Traditional detergent-based decellularization removes the tissue biochemical contents (i.e., glycosaminoglycans). In this new water decellularization protocol, the biochemical contents of cartilage can be preserved. This allows the study of biochemistry and physical content in extracellular matrix as a whole, and this protocol would definitely be useful for studying the effect of tissue microenvironment in supporting chondrocyte growth and functions.

Analysis of Dual Class I Histone Deacetylase and Lysine Demethylase Inhibitor Domatinostat (4SC-202) on Growth and Cellular and Genomic Landscape of Atypical Teratoid/Rhabdoid.

Central nervous system atypical teratoid/rhabdoid tumors (ATRTs) are rare and aggressive tumors with a very poor prognosis. Current treatments for ATRT include resection of the tumor, followed by systemic chemotherapy and radiation therapy, which have toxic side effects for young children. Gene expression analyses of human ATRTs and normal brain samples indicate that ATRTs have aberrant expression of epigenetic markers including class I histone deacetylases (HDAC’s) and lysine demethylase (LSD1). Here, we investigate the effect of a small molecule epigenetic modulator known as Domatinostat (4SC-202), which inhibits both class I HDAC’s and Lysine Demethylase (LSD1), on ATRT cell survival and single cell heterogeneity. Our findings suggest that 4SC-202 is both cytotoxic and cytostatic to ATRT in 2D and 3D scaffold cell culture models and may target cancer stem cells. Single-cell RNA sequencing data from ATRT-06 spheroids treated with 4SC-202 have a reduced population of cells overexpressing stem cell-related genes, including SOX2. Flow cytometry and immunofluorescence on 3D ATRT-06 scaffold models support these results suggesting that 4SC-202 reduces expression of cancer stem cell markers SOX2, CD133, and FOXM1. Drug-induced changes to the systems biology landscape are also explored by multi-omics enrichment analyses. In summary, our data indicate that 4SC-202 has both cytotoxic and cytostatic effects on ATRT, targets specific cell sub-populations, including those with cancer stem-like features, and is an important potential cancer therapeutic to be investigated in vivo.

Development of chitinous nanofiber-based flexible composite hydrogels capable of cell adhesion and detachment

Chitinous nanofibers (NFs) exhibit a specific physiological activity not observed in the powdered form. While three-dimensional cell culture scaffolds are being actively studied, attempts have been made to use chitinous materials as well. In this study, we aimed to develop a cell-adhesive chitinous NF-based flexible hydrogel. Such a gel will be easy to handle, and there is a possibility that the gel will be able to be three-dimensionally molded. For this purpose, the surface of chitinous NFs was modified to introduce vinyl polymerizable functional groups, and the modified NFs were copolymerized with an N-isopropylacrylamide (NIPAM) monomer in an aqueous system. First, by modification of chitosan NFs with maleic anhydride (MA), a flexible gel was obtained by subsequent copolymerization with NIPAM. However, the adhesion of L929 mouse fibroblasts onto MA-modified NFs was extremely lower than that of chitosan NFs, probably due to the simultaneous introduction of anionic groups. The desired cell-adhesive flexible hydrogel was eventually obtained by introducing cationic groups and polymerizable functional groups on the chitinous NF surface. Cells adhered to the gel could be detached by cooling to 4 °C. The composite hydrogel fabricated here may be useful as a cell culture scaffold material in regenerative medicine. Chitinous nanofibers (NFs) exhibit a specific physiological activity not observed in the powdered form. Herein, we aimed to develop a cell-adhesive chitinous NF-based flexible hydrogel. The desired cell-adhesive flexible hydrogel was obtained by introducing cationic groups and vinyl polymerizable functional groups on the surface deacetylated chitin NFs as well as copolymerizing it with N-isopropylacrylamide monomer in an aqueous system. Cells adhered to the gel could be detached by cooling to 4 °C. The composite hydrogel fabricated here may be useful as a cell culture scaffold material in regenerative medicine.

S-protected thiolated hyaluronic acid: In-situ crosslinking hydrogels for 3D cell culture scaffold

The purpose of this study was to synthesize S-protected thiolated hyaluronic acid (HA) and to evaluate its potential for 3D cell culture scaffold. S-protected thiolated HA was synthesized by the covalent attachment of N-acetyl-S-((3-((2,5-dioxopyrrolidin-1-yl)oxy)-3-oxopropyl)thio)cysteine hydrazide ligand to the HA. Hydrogels were characterized for texture, swelling behavior and rheological properties. Furthermore, the potential of S-protected thiolated HA hydrogels as a scaffold for tissue engineering was evaluated by cell proliferation studies with Caco-2 and NIH 3T3 cells. It showed enhanced cohesion upon addition of N-acetyl cysteine (NAC). Dynamic viscosity of S-protected thiolated HA hydrogel was increased up to 19.5-fold by addition of NAC and 10.1-fold after mixing with mucus. Furthermore, Caco-2 and NIH 3T3 cells encapsulated into hydrogels proliferated in-vitro. As this novel S-protected thiolated HA is stable towards oxidation and forms highly cohesive gels when getting into contact with endogenous thiols due to disulfide-crosslinking, it is a promising tool for 3D cell culture scaffold.

Biocompatible Hydrogels Based on Food Gums with Tunable Physicochemical Properties as Scaffolds for Cell Culture.

Hydrogels composed of food gums have gained attention for future biomedical applications, such as targeted delivery and tissue engineering. For their translation to clinical utilization, reliable biocompatibility, sufficient mechanical performance, and tunable structure of polysaccharide hydrogels are required aspects. In this work, we report a unique hybrid polysaccharide hydrogel composed of salecan and curdlan, in which the former is a thickening agent and the latter serves as a network matrix. The physicochemical properties, such as mechanical strength, thermal stability, swelling, and morphology, of the developed composite hydrogel can be accurately modulated by varying the polysaccharide content. Importantly, cytotoxicity assays show the non-toxicity of this hybrid hydrogel. Furthermore, this hydrogel system can support cell proliferation, migration, and function. Altogether, our work proposes a new strategy to build a polysaccharide-constructed hydrogel scaffold, which holds much promise for tissue engineering in terms of cell engraftment, survival, proliferation, and function.

Effect of cell seeding methods on the distribution of cells into the gelatin hydrogel nonwoven fabric.

Recently, as one trial to enhance the biological functions of cells, several attempts have been performed to allow cells to interact in a three dimension (3D) manner. To do that, it is of prime importance to achieve a homogeneous distribution and proliferation of cells in 3D scaffolds, which affects the biological functions of cells [1]. The researches of 3D cell culture scaffolds have been extensively reported while several materials with different inner structures have been prepared from synthetic polymers, natural polymers, and bioceramics [2]. There have been reported on 3D scaffolds with various inner structures [3], such as sponge [4], 3D printer structure [5], fiber (including nanofiber) [6], woven fabric [7], non-woven fabric [8], and a braid [9]. Among them the effect of seeding methods on the cell distribution has been evaluated to hydrogel scaffolds [10,11]. For the hydrogel scaffolds reported so far, the mechanical property is too poor to allow cells to seed homogeneously in the scaffolds [12]. As a trial to break through the issue, the hydrogel of gelatin non-woven fabrics has been explored to demonstrate a homogeneous proliferation of cells in the non-woven fabric [13]. This study is undertaken to evaluate the effect of cell seeding methods on the proliferation and distribution of cells in the gelatin hydrogel non-woven fabrics (GHNF). We examine the proliferation behavior of cells and their membrane damage based on lactate dehydrogenase (LDH) activity after different cell seedings.

Hydrogel formation by short D-peptide for cell-culture scaffolds.

The present study reports that a short oligopeptide D-P1, consisting of only five D-amino acids, self-assembled into entangled nanofibers to form a hydrogel that functioned as a scaffold for cell cultures. D-P1 (Ac-D-Phe-D-Phe-D-Phe-Gly-D-Lys) gelated aqueous buffer solution and water at a minimum gelation concentration of 0.5wt%. The circular dichroism (CD) measurements demonstrated the formation of a β-sheet structure in the self-assembly of D-P1. We investigated the gelation properties and CD spectra of both the D- and L-forms of the oligopeptide, and found only a minimal difference between them. The D-P1 hydrogel was resistant to a protease, whereas the L-P1 hydrogel was rapidly degraded. Both oligopeptides exhibited nontoxic properties to human cancer cells and embryoid bodies (EBs) derived from human-induced pluripotent stem cells. Additionally, we succeeded in forming spheroids of HeLa cells on the D-P1 hydrogel, which indicates the potential of this hydrogel for 3-dimensional cell culture.

Dynamic Covalent C═C Bond, Cross-Linked, Injectable, and Self-Healable Hydrogels via Knoevenagel Condensation.

Hydrogels have a wide range of applications in the fields of biomedicine, flexible electronics, and bionics. In this study, injectable and self-healable hydrogels were first prepared based on a dynamic covalent C═C bond formed via the Knoevenagel condensation reaction between poly(ethylene glycol) dicyanoacetate and water-soluble poly(vanillin acrylate). Three kinds of catalysts (phosphate buffer, zeolitic imidazolate framework-8, and tertiary amine) were used in Knoevenagel condensation for preparing hydrogels. All hydrogels in this study could be formed in situ, and their gelation time ranged from seconds to minutes. The properties and application of hydrogels could be customized according to the type of catalyst employed. 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) results indicated that all the components and hydrogels exhibited low toxicity, and the hydrogels could be used as 3D cell culture scaffolds. Because of the dynamic covalent C═C bond formed by Knoevenagel condensation, the resultant hydrogels were found to be dynamic and showed good self-healing properties. This work presents a new dynamic covalent chemistry for the preparation of self-healable materials.

Homing of adipose stem cells on the human amniotic membrane as a scaffold: A histological study.

BackgroundThe human amniotic membrane (HAM) is a suitable and effective scaffold for cell culture and delivery, and adipose-derived stem cells (ADSCs) are an important source of stem cells for transplantation and chondrogenic differentiation.ObjectiveTo assess the practicability of a cryopreserved HAM as a scaffold in cell proliferation and differentiation in vitro.Materials and MethodsIn this experimental study, adipose tissue samples were harvested from the inguinal region of male patients aged 15-30 years. Flow cytometry was used to identify CD31, CD45, CD90, and CD105 markers in adipose stem cells. HAM was harvested from donor placenta after cesarean section, washed, trypsin-based decellularized trypsinized decellularized, and used as a scaffold via three methods: 1) ADSCs were differentiated into chondrocytes on cell culture flasks (monolayer method), and after 14 days of culture, the cells were transferred and cultured on both sides of the HAM; 2) ADSCs were cultured and differentiated directly on both sides of the HAM for 14 days (scaffold-mediated differentiation); and 3) chondrocytes were differentiated with micromass culture for 14 days, transferred on HAM, and tissue slides were histologically analyzed qualitatively.ResultsFlow cytometry confirmed the presence of mesenchymal stem cells. Histological findings revealed that the cells adhered and grew well on the stromal layer of HAM. Among the three methods, scaffold-mediated differentiation of ADSCs showed the best results.Conclusion ADSCs have excellent attachment, viability, and differentiation capacity in the stromal side of HAM. Additionally, the direct culture and differentiation of ADSCs on HAM is more suitable than the culture of differentiated cells on HAM.

Preparation and Characterization of Thermoresponsive PEG-Based Injectable Hydrogels and Their Application for 3D Cell Culture.

We report here the synthesis of a series of ethylene glycol-based triblock copolymers containing a hydrophilic middle segment of poly(ethylene glycol) methyl ether methacrylate (PEGMA) and two temperature-responsive segments of diethylene glycol methyl ether methacrylate (DEGMA) at both ends via the reversible addition-fragmentation chain-transfer (RAFT) polymerization. While the corresponding temperature-responsive homopolymer (PDEGMA) and the diblock copolymer (PDEGMA-b-PPEGMA) could not form a gel, the triblock copolymers (PDEGMA-b-PPEGMA-b-PDEGMA) could form a physical gel at certain concentrations and at temperatures above the lower critical solution temperature (LCST). This sol-gel transition is fully reversible and can be repeated several times. Depending on the chain length of the middle block and two end blocks, a physical gel could be formed at a minimum polymer concentration of 5 wt %. In addition, a mechanically strong gel could be easily formed within 5 s at the maximum concentration of 20 wt % and at a temperature of 37 °C. Considering the good cell compatibility and soft rubbery nature of the triblock copolymers, they can potentially be used as injectable scaffold for cell culture and tissue engineering applications.

Succinoglycan dialdehyde-reinforced gelatin hydrogels with toughness and thermal stability

Pure gelatin hydrogel (PG) has limited practical applications due to their thermal instability and unfavorable mechanical properties. To overcome these limitations, dually crosslinked hydrogels were developed by imparting chemical crosslinking to existing physically crosslinked gelatin hydrogel networks using succinoglycan dialdehyde (SGDA) as a macromolecular crosslinker. SGDA-reinforced gelatin hydrogels (SGDA/Gels) displayed an 11 times higher compressive stress under identical deformation strain and a 1040% improvement in storage modulus (G′) than PG. In addition, chemical crosslinking induced by SGDA increased the thermal stability of SGDA/Gels, such that they did not decompose at 60 °C, as confirmed by oscillatory temperature ramp experiments. The newly synthesized SGDA/Gels with reinforced networks and thermal stability exhibit potential for long-term use as controlled drug delivery carriers and 3D cell culture scaffolds for tissue engineering.

Synthesis and characterization of thiol-acrylate hydrogels using a base-catalyzed Michael addition for 3D cell culture applications.

There is significant interest in developing new approaches for culturing mammalian cells in a three-dimensional (3D) environment due to the fact that it better recapitulates the in vivo environment. The goal of this work was to develop thiol-acrylate, biodegradable hydrogels that possess highly tunable properties to support in vitro 3D culture. Six different hydrogel formulations were synthesized using two readily available monomers, a trithiol (ETTMP 1300 [ethoxylated trimethylolpropane tri(3-mercaptopropionate) 1300]) and a diacrylate (PEGDA 700 [polyethylene glycol diacrylate 700]), polymerized by a base-catalyzed Michael addition reaction. The resultant hydrogels were homogeneous, hydrophilic, and biodegradable. Different mechanical properties such as gelation time, storage modulus (or the elasticity G'), swelling ratio, and rate of degradation were tuned by varying the weight percentage of polymer, the molar ratio of thiol-to-acrylate groups, and the pH of the solution. Cytocompatibility was assessed using two model breast cancer cell lines by both 2D and 3D cell culturing approaches. The hydrogel formulations with a thiol-to-acrylate molar ratio of 1.05 were found to be optimal for both 2D and 3D cultures with MDA-MB-231 cellular aggregates found to be viable after 17 days of 3D continuous culture. Finally, MCF7 cells were observed to form 3D spheroids up to 600 μm in diameter as proof of principle for the thiol-acrylate hydrogel to function as a scaffold for in vitro 3D cell culture. A comparison of the different mechanical properties of the six hydrogel formulations coupled with in vitro cell culture results and findings from previously published hydrogels conclude that the thiol-acrylate hydrogels have significant potential as a scaffold for 3D cell culture.

3D bioprinting of tissue engineering scaffold for cell culture

PurposeThe 3 D bioprinting technology is used to prepare the tissue engineering scaffold with precise structures for the cell proliferation and differentiation.Design/methodology/approachAccording to the characteristics of the ideal tissue engineering scaffold, the microstructural design of the tissue engineering scaffold is carried out. The bioprinter is used to fabricate the tissue engineering scaffold with different structures and spacing sizes. Finally, the scaffold with good connectivity is achieved and used to cell PC12 culture.FindingsThe results show that the pore structure with the line spacing of 1 mm was the best for cell culture, and the survival rate of the inoculated cells PC12 is as high as 90%. The influence of the pore shape on the cell survival is not evidence.Originality/valueThis study shows that tissue engineering scaffolds prepared by 3 D bioprinting have graded structure for three-dimensional cell culture, which lays the foundation for the later detection of drug resistance.

Electrohydrodynamic jet 3D printing of PCL/PVP composite scaffold for cell culture

Controlled printing of biodegradable and bioresorbable polymers at desired 3D scaffold is of great importance for cell growth and tissue regeneration. In this work, a novel electrohydrodynamic jet 3D printing technology with the resultant effect of electrohydrodynamic force and thermal convection was developed, and its feasibility to fabricate controllable filament composite scaffolds was verified. This method introduces an effective thermal field under the needle to simultaneously enhance the ink viscosity, jetting morphology controllability and printing structure solidify. The fabrication mechanisms of thermal convection on jetting morphology and printed structures feature were investigated through theoretical analysis and experimental characterization. Under optimized conditions, a stable and finer jet was formed; then with the use of this jet, various 3D structures were directly printed at a high aspect ratio ~30. Furthermore, the PCL/PVP composite scaffolds with the controllable filament diameter (~10 μm) which is closed to living cells were printed. Cell culture experiments showed that the printed scaffolds had excellent cell biocompatibility and facilitated cellular proliferation in vitro. It is a great potential that the developed electrohydrodynamic jet 3D printing technology might provide a novel approach to directly print composite synthetic biopolymers into flexibly scale structures for tissue engineering applications.

The Efficacy of Electrospun PAN/Kefiran Nanofiber and Kefir in Mammalian Cell Culture: Promotion of PC12 Cell Growth, Anti-MCF7 Breast Cancer Cells Activities, and Cytokine Production of PBMC.

BackgroundKefiran is a useful polysaccharide made of branched glucogalactose which is produced by microorganisms. Here the anti-MCF-7 breast cancer cells activity of kefiran and cytokine productions (IL-6) of peripheral blood mononuclear cells (PBMC) treated by kefiran was studied. Also, the effect of using kefiran as a useful and cost-effective scaffold in neural stem cell culture (PC12 cell culture) was investigated.Material and MethodsKefiran was produced from raw milk with 0.5% fat and 10 g of kefir grains. After incubation for 48 hrs at room temperature, the solvent collected (crude kefiran). These samples were kept at 100°C for 1 hr (boiled kefiran) and the supernatant was precipitated by ethanol (pure kefiran). Then, the electrospun nanofibers, pure polyacrylonitrile (PAN), PAN/kefiran 5%, and PAN/kefiran 10% were fabricated and used as scaffolds in the cell culture. The structure of fabricated was studied by SEM and the cytokine production (IL-6) in vitro in the cell culture supernatant of PBMC line after treatment with kefiran (1mg/mL, 5 mg/mL) and kefiran-PAN 5% and 10% were carried out by enzyme-linked immunosorbent assay (ELISA). The attachment of PC12 cells was examined by inverted microscope. Also, cytotoxicity of kefiran for PC12 and MCF7 cells and morphological changes of PC12 cells were evaluated by MTT and Cresyl violet staining (Nissl staining) respectively.ResultsThe mean diameter of fabricated PAN/kefiran 5% and 10% nanofibers were 310.2±43.97 nm. The contact angle measurement results (26.9± 1.9 for the pure PAN scaffold vs 12.3± 1.13 for the PAN/kefiran) revealed enhanced hydrophilicity of scaffolds upon the incorporation of kefiran and PAN. Seeding of PC12 cells on the scaffolds showed that fabrication of kefiran into PAN led to the enhancement of cell attachment, proliferation, and morphological changes. Also, the promotion of PBMC growth and decreasing of MCF7 cell lines viability were shown through MTT assay. No significant changes were measured for the level of IL-6 in PAN/kefiran 5% treated cells compared to the control (p ≥ 0.05).ConclusionThese results suggest superior properties of kefiran/PAN nanofibrous scaffolds for the neural stem cell culture especially for repairing injured spinal cord. Also, the pure kefiran could be used for the enhancement of PBMC growth and reducing the MCF7 cancerous cells growth. So, using biocompatible, anti-bacterial, and anti-tumor kefiran/PAN nanofibers for regenerative medicine seems promising.

Bioengineering tools to speed up the discovery and preclinical testing of vaccines for SARS-CoV-2 and therapeutic agents for COVID-19.

This review provides an update for the international research community on the cell modeling tools that could accelerate the understanding of SARS-CoV-2 infection mechanisms and could thus speed up the development of vaccines and therapeutic agents against COVID-19.Many bioengineering groups are actively developing frontier tools that are capable of providing realistic three-dimensional (3D) models for biological research, including cell culture scaffolds, microfluidic chambers for the culture of tissue equivalents and organoids, and implantable windows for intravital imaging.Here, we review the most innovative study models based on these bioengineering tools in the context of virology and vaccinology. To make it easier for scientists working on SARS-CoV-2 to identify and apply specific tools, we discuss how they could accelerate the discovery and preclinical development of antiviral drugs and vaccines, compared to conventional models.

Synthesis of spiropyran with methacrylate at the benzopyran moiety and control of the water repellency and cell adhesion of its polymer film.

Stimuli-responsive materials have been actively researched over the past few decades. Among such materials, spiropyran is one of the most attractive compounds because the structure and polarity of the material are dramatically changed after photo irradiation, unlike other materials. In this work, we designed and synthesized a spiropyran derivative (SpMA) with a methacryloyl group on the nitrobenzene ring of a spiropyran skeleton. The UV spectra of the newly synthesized SpMA showed the photo-isomerization of spiropyran. The maximum absorption wavelength (λmax) of SpMA was 616 nm in n-hexane, a nonpolar solvent, although λmax of SpMA was 532 nm in methanol, a polar protic solvent, which resulted in an 84 nm blue-shift. SpMA was successfully polymerized by ruthenium (Ru)-catalyzed living radical polymerization. Poly(SpMA) (PSpMA) was then spin-coated on a PET substrate in order to control the surface properties of water repellency and cell adhesion. The water repellency was decreased approximately 10° under UV irradiation, because of the polarity change of PSpMA caused by photo-isomerization from the spiropyran (SP) type to the merocyanine (MC) type. In addition, NIH3T3 cells were spread only on 6% of the surface of the PSpMA thin film after UV irradiation compared with no UV irradiation. The polarity change of PSpMA by photo-isomerization is also believed to be the reason for this behavior. As a result, we successfully synthesized a photo-controllable cell culture scaffold.

PDGFBB-modified stem cells from apical papilla and thermosensitive hydrogel scaffolds induced bone regeneration

Bone defects caused by cancer surgery or trauma have a strong negative impact on human health. Treatment with cell and material-based complexes provides an alternative strategy for the regeneration of damaged bone tissue. The good physical properties and suitable biodegradability of a thermosensitive hydrogel has been shown to act as a valuable scaffold. Platelet derived growth factor BB (PDGFBB) is mainly secreted by platelets and promotes the migration and angiogenesis of mesenchymal stem cells (MSCs). Although PDGFBB is known to indirectly enhance bone repair in vivo, the effects of PDGFBB on stem cells from apical papilla (SCAPs) require further investigation. In our study, the proliferation of cells was investigated by the cell counting kit-8 and live/dead staining methods. The results indicated that PDGFBB promoted the proliferation of SCAPs. Real-time polymerase chain reaction and Western blot experiments were used to detect osteogenic genes and proteins. Moreover, calvarial defects were created in Sprague-Dawley rats and different complexes implanted. The results shown by micro-CT and hematoxylin and eosin analysis demonstrated that the hydrogel combined with lentiviral supernatant-green fluorescent protein-PDGFBB significantly improved new bone formation and mineralization compared with the other three groups. In summary, our research showed that a thermosensitive hydrogel can be used as a scaffold for 3D cell culture, and PDGFBB gene-modified SCAPs can improve bone formation in calvarial defects.

Development of rolled scaffold for high-density adherent cell culture.

Bioreactors for large-scale culture of mammalian cells are playing vital roles in biotechnology and bioengineering. Various bioreactors have been developed, but their capacity and efficiency are often limited by insufficient mass transfer rate and high shear stress. A rolled scaffold (RS) is a fully defined scaffold for high-density adherent culture of mammalian cells. The RS is a polymer film with spacers, that is rolled into a cylinder with a pre-determined gap between each turn. Cells are cultured on its inner surfaces, while media flows through the gap. The RS exhibits high surface-area-to-volume ratio over 100cm2/mL and can transport nutrients and gases with significantly reduced shear stress via convection in a unidirectional laminar flow, rather than diffusion and random turbulent flow as in stirred-tank bioreactors. In this paper, we expanded Chinese Hamster Ovary cells with RS bioreactors and demonstrated cell culture density over 60 million cells/mL with a growth rate higher than conventional suspension culture. Besides, murine embryonic stem cells were successfully expanded without losing their pluripotency. The RS will provide an affordable, scalable, and reliable platform for large-scale culture of recombinant cells in biopharmaceutical industries and shear-sensitive stem cells for tissue engineering.