Gel beads composed of collagen reconstituted in alginate

Spherical gel beads of collagen/alginate were prepared by discharging droplets of a mixture containing collagen (1.07-1.9 mg/ml) and alginate (1.2-1.5% w/v) into 1.5% w/v CaCl2 solution at 4°C. Collagen in the gel beads was reconstituted by raising the temperature to 37°C after alginate was liquefied by citrate. Scanning electron microscopy of the beads revealed the characteristic fibrous structure of collagen. To demonstrate the application of this new technique in cell culture, GH3 rat pituitary tumor cells were entrapped and grown in the gel beads. The immobilized cells proliferated to a density of 1.95 x 106 cell/ml which is about an order of magnitude higher than that grown in the alginate beads.     

Calcium-alginate gel bead cross-linked with gelatin as microcarrier for anchorage-dependent cell culture

Valuable products obtainedfrom the cultivation of anchorage-dependent mammalian cells require large-scale processes to obtain commercially useful quantities. It is generally accepted that suspension culture is the ideal mode of operation. Because anchorage-dependent cells need surfaces to be able to attach and spread, the incorporation of microcarriers to suspension culture is indispensable. Since the dextran-based microcarrier wasfirst introduced, many different types of microcarriers have been developed and commercialized. In this study, alginate-based microcarriers were made in the following order: (i) calcium-alginate gel beads prepared by dropping a blend of sodium alginate and propylene glycol alginate (PGA) into calcium chloride solution, (ii) the PGA section of gel beads cross-linked with gelatin in alkaline solution (i.e., via the transacylation reaction between the ester group of PGA and amino group of gelatin), and (iii) gelatin membrane around the beads further cross-linked by glutaraldehyde. The glutaraldehyde-treated gelatintransacylated PGA/alginate microcarrier showed superior features in high stability under phosphate-containing solution, density close to that of culture medium, and transparency. Moreover, the Chinese hamster ovary CHO-KI and amphotropic retrovirus producer PA317 cells cultivated on the newly synthesized microcarriers exhibited similar growth kinetics of these two types of cell lines cultured on commercial polystyrene microcarriers. However, cell morphology was easily monitored on the transparent microcarriers made in this study.     

Serial propagation of mammalian cells on gelatin-coated microcarriers

The ability to serially propagate mammalian cells in microcarrier cultures is essential for large-scale operation. The success of such serial propagation depends on viable dissociation of cells from microcarriers and the normal growth and product formation after subsequent reinoculation. The high pH treatment developed for dissociating cells from DEAE-derivatized microcarriers was not as effective for a number of cell strains cultivated on gelatin-coated microcarriers. By prewashing the cell-laden microcarriers with buffer containing a chelating agent, bovine kidney cells, BK, human embryonic foreskin fibroblasts, FS-4, and continuous human kidney cells, TCL-598 which produces prourokinase, were viably dissociated from commercially available gelatin-coated microcarriers, Cytodex-3. Cells dissociated from microcarriers reattached and grew on micro-carriers subsequent to inoculation into subcultures. However, after subculturing, cells may attach at different rates to newly added beads and to conditioned microcarriers which cells had previously grown. It resulted in an uneven cell distribution on microcarriers and inferior growth kinetics. This effect was more profound for BK and FS-4 cells which are propagated with a low multiplication ratio. Specifically, BK cells attach to conditioned beads at a faster rate than to new beads, while FS-4 cells attach to new beads faster than to conditioned beads. Thus, for these two cell strains, a separator was used to separate the microcarriers from the suspension of dissociated cells before subsequent inoculation. For TCL-598 cells, which are propagated at a high multiplication ratio, this dissociation technique can be applied directly without the separation of dissociated cells and conditioned microcarriers. All the three cell lines tested exhibit normal growth kinetics in serial propagation on microcarriers. Furthermore, the production of prourokinase by TCL598 cells serially propagated on microcarriers was comparable to that inoculated from roller bottles.     

Growth of endothelial and HeLa cells on a new multipurpose microcarrier that is positive,negative or collagen coated

A new cell culture microcarrier that can be covalently bonded by cell attachment proteins and can be thin-sectioned for electron microscopy was synthesized. It was easily made by sulfonating cross-linked polystyrene beads for a negative surface charge followed by covalent attachment of polyethylenimine for a positive charge. Cell attachment proteins, e.g. collagen, was covalently bonded directly to the microcarrier using a carbodiimide or after activating the microcarrier surface with glutaraldehyde. HeLa-S3 cells attached, spread and grew to confluence more efficiently on the positive microcarriers and those coated with collagen than on the negative ones. Endothelial cells grew best on those with a negative surface charge. The nature of the microcarrier surface was not the only aspect involved in cell adhesion but also the type of serum proteins adsorbed. Qualitatively different proteins coated the microcarriers depending upon whether the carrier was negative, positive or coated with collagen. Comparison of various types of available microcarriers indicated that the modified cross-linked polystyrene beads used here were best for transmission and scanning electron microscopy. Endothelial cells grown on the microcarriers had the same ultrastructure as cells grown in monolayers in culture dishes. Of a variety of microcarriers tested the modified cross-linked polystyrene beads were the only ones that could be used for both ultrastructural and biochemical techniques.     

Fabrication of artificial endothelialized tubes with predetermined three-dimensional configuration from flexible cell-enclosing alginate fibers

One possible strategy for creating three-dimensional (3D) tissue-engineered organs in vitro is to develop a vasculature for sufficient transport of oxygen and nutrients within these constructs. Here, we describe a novel technique to fabricate endothelialized tubes with predetermined 3D configuration, as a starting point for self-developing capillary-like networks in vitro. Calcium-alginate hydrogel fibers of ca. 250 and 500 mum in diameter, enclosing bovine carotid artery vascular endothelial cells (BECs), were used as templates for endothelialized tubes. Fibers were prepared by extruding a 2% (w/v) sodium alginate solution containing BECs into a 100 mM calcium chloride solution flowing in the same direction. Fibers were embedded in type I collagen gels and enzymatically degraded by alginate lyase, resulting in channels with predetermined 3D configuration filled with a BEC suspension. Cells attached to and covered the surfaces of the channels. Exposing the cells to medium containing basic fibroblast growth factor resulted in their migration into the ambient collagen gel and self-assembly into capillary-like structures. These results demonstrate that using artificial endothelialized tubes with predetermined 3D configuration, as a starting point for a self-developing capillary-like network, could be potentially useful for constructing 3D tissue-engineered organs.     

The role of collagen structure in mitogen stimulation of ERK,cyclin D1 expression,and G1-S progression in rat hepatocytes

Adhesion to type 1 collagen can elicit different cellular responses dependent upon whether the collagen is in a fibrillar form (gel) or monomeric form (film). Hepatocytes adherent to collagen film spread extensively, express cyclin D1, and increase DNA synthesis in response to epidermal growth factor, whereas hepatocytes adherent to collagen gel have increased differentiated function, but lower DNA synthesis. The signaling mechanisms by which different forms of type I collagen modulate cell cycle progression are unknown. When ERK MAP kinase activation was analyzed in hepatocytes attached to collagen film, two peaks of ERK activity were demonstrated. Only the second peak, which correlated with an increase of cyclin D1, was required for G1-S progression. Notably, this second peak of ERK activity was absent in cells adherent to collagen gel, but not required in the presence of exogenous cyclin D1. Expression of activated mutants of the Ras/Raf/MEK signaling pathway in cells adherent to collagen gel restored ERK phosphorylation and DNA synthesis, but differentially affected cell shape. Although Ras, Raf, and MEK all increased expression of cyclin D1 on collagen film, only Ras and Raf significantly up-regulated cyclin D1 levels on collagen gel. These results demonstrate that adhesion to polymerized collagen induces growth arrest by inhibiting the Ras/ERK-signaling pathway to cyclin D1 required in late G1.     

The effect of immobilization on recombinant protein production in plant cell culture

The effects of encapsulation on the production of recombinant human proteins by Nicotiana tabacum cells were investigated using alginate, carrageenan, and agar as immobilization matrices. Experiments showed that cell encapsulation in alginate increased the production of human granulocyte-macrophage colony-stimulating factor (GM-CSF) in tobacco cells by approximately 50%. Alginate also yielded the highest quality beads and the most reproducible growth results. The most likely cause for this increased protein production is the altered growth conditions within the alginate beads resulting in a prolonged exponential growth phase. To characterize these effects, we compared growth performance and protein production for various gel geometries, bead sizes, and volume fractions of beads.     

Type I collagen structure regulates cell morphology and EGF signaling in primary rat hepatocytes through cAMP-dependent protein kinase A

Adhesion to type 1 collagen elicits different responses dependent on whether the collagen is in fibrillar (gel) or monomeric form (film). Hepatocytes adherent to collagen film spread and proliferate, whereas those adherent to collagen gel remain rounded and growth arrested. To explore the role of potential intracellular inhibitory signals responsible for collagen gel-mediated growth arrest, cAMP-dependent protein kinase A (PKA) was examined in hepatocytes adherent to collagen film or gel. PKA activity was higher in hepatocytes on collagen gel than on film during G1 of the hepatocyte cell cycle. Inhibition of PKA using H89 increased cell spreading on collagen gel in an EGF-dependent manner, whereas activation of PKA using 8-Br-cAMP decreased cell spreading on collagen film. PKA inhibition also restored ERK activation, cyclin D1 expression and G1-S progression on collagen gel, but had no effect on cells adherent to collagen film. Analysis of EGF receptor phosphorylation revealed that adhesion to collagen gel alters tyrosine phosphorylation of the EGF receptor, leading to reduced phosphorylation of tyrosine residue 845, which was increased by inhibition of PKA. These results demonstrate that fibrillar type 1 collagen can actively disrupt cell cycle progression by inhibiting specific signals from the EGF receptor through a PKA-dependent pathway.     

Survival of Bifidobacterium longum Immobilized in Calcium Alginate Beads in Simulated Gastric Juices and Bile Salt Solution

Bifidobacterium longum KCTC 3128 and HLC 3742 were independently immobilized (entrapped) in calcium alginate beads containing 2, 3, and 4% sodium alginate. When the bifidobacteria entrapped in calcium alginate beads were exposed to simulated gastric juices and a bile salt solution, the death rate of the cells in the beads decreased proportionally with an increase in both the alginate gel concentration and bead size. The initial cell numbers in the beads affected the numbers of survivors after exposure to these solutions; however, the death rates of the viable cells were not affected. Accordingly, a mathematical model was formulated which expressed the influences of several parameters (gel concentration, bead size, and initial cell numbers) on the survival of entrapped bifidobacteria after sequential exposure to simulated gastric juices followed by a bile salt solution. The model proposed in this paper may be useful for estimating the survival of bifidobacteria in beads and establishing optimal entrapment conditions.     

Survival of Bifidobacterium longum immobilized in calcium alginate beads in simulated gastric juices and bile salt solution

Bifidobacterium longum KCTC 3128 and HLC 3742 were independently immobilized (entrapped) in calcium alginate beads containing 2, 3, and 4% sodium alginate. When the bifidobacteria entrapped in calcium alginate beads were exposed to simulated gastric juices and a bile salt solution, the death rate of the cells in the beads decreased proportionally with an increase in both the alginate gel concentration and bead size. The initial cell numbers in the beads affected the numbers of survivors after exposure to these solutions; however, the death rates of the viable cells were not affected. Accordingly, a mathematical model was formulated which expressed the influences of several parameters (gel concentration, bead size, and initial cell numbers) on the survival of entrapped bifidobacteria after sequential exposure to simulated gastric juices followed by a bile salt solution. The model proposed in this paper may be useful for estimating the survival of bifidobacteria in beads and establishing optimal entrapment conditions.     

Factors affecting cell attachment, spreading, and growth on derivatized microcarriers. I. Establishment of working system and effect of the type of the amino-charged groups

A working system for studying the effects of factors involved in the chemical nature of microcarriers on cell attachment, spreading, and growth was established. The system is based on polyacrylamide beads, prepared by the emulsion polymerization technique. Sieved beads of desirable mean diameter were derivatized to generate controlled amounts of primary and tertiary amino groups. These microcarriers were used for the propagation of four different cell strains: BHK, MDCK, CEF, and MRC-5. It was found that BHK cells attach and spread significantly faster on primary amino-derivatized beads than those with tertiary amino groups, and at a lower degree of charging. Cell yields of MDCK cells (with pronounced epithelial morphology) propagated on primary amino-derivatized beads were higher than that obtained for the tertiary amino-derivatized microcarriers. On the other hand, CEF and MRC-5 cells (with pronounced fibroblast morphology) achieved higher cell yields on the tertiary amino-derivatized microcarriers.     

Growth of recombinant fibroblasts in alginate microcapsules

To develop a novel strategy of nonautologous somatic gene therapy, we now demonstrate the feasibility of culturing genetically modified fibroblasts within an immunoprotective environment and the optimal conditions required for their continued survival in vitro. When mouse Ltk(-) fibroblasts transfected with the human growth hormone gene were enclosed within permselective microcapsules fabricated from alginate-polylysine-alginate, they continued to secrete human growth hormone at the same rates as the nonencapsulated cells. They also continued to proliferate in vitro for at least 1 month even though their viability gradually declined to about 50%. The viability can be improved by controlling for (a) temperature during encapsulation, (b) duration of treatment with polylysine, (c) duration of liquefying the core alginate with sodium citrate, and (d) cell density at the time of encapsulation. The best conditions leading to improved survival and maximum proliferation of cells within the microcapsules were obtained by encapsulating the cells at 4 to 10 degrees C instead of room temperature, coating the microspheres with polylysine for 6 to 10 min instead of 20 min, liquefying the core alginate by treating with citrate for 20 min instead of 6 to 10 min, and using a concentration of 2 x 10(6) cells/mL of alginate for encapsulation. Under such conditions, normally adherent and genetically engineered mouse fibroblasts survived and proliferated optimally within the microcapsule environment. The encapsulated fibroblasts maintained their level of transgene expression while recombinant gene products such as human growth hormone could diffuse through the microcapsule membrane without impediment. The demonstration that genetically modified fibroblasts can survive and continue to deliver recombinant gene products from within these microcapsules and the optimization for their maximal viability and growth within microcapsules should increase the potential for success in using such microencapsulated recombinant cells for somatic gene therapy. (c) 1994 John Wiley & Sons, Inc.     

Scalable culture and cryopreservation of human embryonic stem cells on microcarriers

As a result of their pluripotency and potential for unlimited self‐renewal, human embryonic stem cells (hESCs) hold tremendous promise in regenerative medicine. An essential prerequisite for the widespread application of hESCs is the establishment of effective and efficient protocols for large‐scale cell culture, storage, and distribution. At laboratory scales hESCs are cultured adherent to tissue culture plates; these culture techniques are labor‐intensive and do not scale to high cell numbers. In an effort to facilitate larger scale hESC cultivation, we investigated the feasibility of culturing hESCs adherent to microcarriers. We modified the surface of Cytodex 3 microcarriers with either Matrigel or mouse embryonic fibroblasts (MEFs). hESC colonies were effectively expanded in a pluripotent, undifferentiated state on both Matrigel‐coated microcarriers and microcarriers seeded with a MEF monolayer. While the hESC expansion rate on MEF‐microcarriers was less than that on MEF‐plates, the doubling time of hESCs on Matrigel‐microcarriers was indistinguishable from that of hESCs expanded on Matrigel‐coated tissue culture plates. Standard hESC cryopreservation methodologies are plagued by poor viability and high differentiation rates upon thawing. Here, we demonstrate that cryopreservation of hESCs adherent to microcarriers in cryovials provides a higher recovery of undifferentiated cells than cryopreservation of cells in suspension. Together, these results suggest that microcarrier‐based stabilization and culture may facilitate hESC expansion and storage for research and therapeutic applications. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Development of an optical system for the non‐invasive tracking of stem cell growth on microcarriers

The emergence of medicinal indications for stem cell therapies has seen a need to develop the manufacturing capacity for adherent cells such as mesenchymal stem cells (MSCs). One such development is in the use of microcarriers, which facilitate enhanced cell densities for adherent stem cell cultures when compared with 2D culture platforms. Given the variety of stem cell expansion systems commercially available, novel methods of non‐invasive and automated monitoring of cell number, confluence, and aggregation, within disparate environments, will become imperative to process control, ensuring reliable and consistent performance. The in situ epi‐illumination of mouse embryonic fibroblasts and human mesenchymal stem cells attached to Cytodex 1 and 3 microcarriers was achieved using a bespoke microscope. Robust image processing techniques were developed to provide quantitative measurements of confluence, aggregate recognition, and cell number, without the need for fluorescent labeling or cell detachment. Large datasets of cells counted on individual microcarriers were statistically analyzed and compared with NucleoCounter measurements, with an average difference of less than 7% observed from days 0 to 6 of a 12‐day culture noted, prior to the onset of aggregation. The developed image acquisition system and post‐processing methodologies were successfully applied to dynamically moving colonized microcarriers. The proposed system offers a novel method of cell identification at the individual level, to consistently and accurately assess viable cell number, confluence, and cell distribution, while also minimizing the variability inherent in the current invasive means by which cells adhered to microcarriers are analyzed. Biotechnol. Bioeng. 2017;114: 2032–2042. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

大规模动物细胞培养技术研究进展

利用动物细胞大规模培养技术可生产多种生物制品,为提高细胞活力和表达水平及有利于表达产物的纯化,采用有多种添加成分的无血清培养基培养细胞,选择更有利于细胞生长又可提高培养细胞密度的微载体和条件温和、易操作、气体交换速度快的生物反应器,在线监控细胞生存环境和生理活动,减少培养过程培养基中的抑制因素,可创造更适合细胞生存的环境,提高表达水平,向细胞中导入抗凋亡基因,可提高细胞活性和蛋白产量。利用多也微载体以球转球方式大规模培养动物细胞有很好的发展前景。     

Stability of alginate-immobilized algal cells

Investigations were carried out using immobilized Chlorella cells to determine the diameter, compressibility, tolerance to phosphate chelation, and ability to retain algal cells during incubation of various alginate beads. These physical bead characteristics were found to be affected by a variety of interactive factors, including multivalent cation type (hardening agent) and cell, cation, and alginate concentration, the latter exhibiting a predominant influence. The susceptibility of alginate beads to phosphate chelation was found to involve a complex interaction of cation type, concentration, and pH of phosphate solution. A scale of response ranging from gel swelling to gel shrinking was observed for a range of conditions. However, stable calcium alginate beads were maintained in incubation media with a pH of 5.5 and a phosphate concentration of 5muM. A preliminary investigation into cell leakage from the beads illustrated the importance of maintaining a stable gel structure and limiting cell growth to reduce leakage.     

Platelet-Derived Growth Factors-BB and Fibroblast Growth Factors-Base Induced Proliferation of Schwann Cells in a 3D Environment

The proliferation of neonatal Schwann cells (SCs) in response to mitogenic agents has been well analyzed in vitro (mono-layer-culture method, 2D environment), but not in vivo (3D environment). To assess the mitogenic effect of platelet-derived growth factors-BB (PDGF-BB), Fibroblast Growth Factors-base (bFGF), and their combinations for SCs in collagen gel (three-dimensional, 3D environment), we have developed an integrated microfluidic device on which can reproducibly measure the proliferation from small number of cells (1–100). The rat SCs were cultured for 4 week at the different concentrations of growth factors generated by concentration gradient generator. In the collagen gel culture, almost all of the cells in colonies presented a round cell morphology and maintained their round morphology by the 4th week. The results showed that PDGF-BB and bFGF are all capable of moderately stimulating SCs growth and every group reached the peak in the growth curve at 3 weeks. Moreover, the proliferation test using the conventional method was performed simultaneously and revealed similar results. The biggest difference between 2D and 3D was that cells decrease more remarkable in 3D than that in 2D at 4 weeks. And at 2 and 3 weeks, the growth rate in the collagen gel with 7.14/2.86 and 8.57/1.43 ng/mL groups was higher than that in the mono-layer culture. Our results showed that PDGF-BB and bFGF are capable of moderately stimulating neonatal SCs growth, respectively and synergistically, and the microfluidic technique is highly controllable, contamination free, fully automatic, and inexpensive.     

Pellet culture elicits superior chondrogenic redifferentiation than alginate‐based systems

Although pellet culture and encapsulation of chondrocytes into gel‐like biomaterials have lead to major advances in cartilage tissue engineering, a quantitative comparative characterization of cellular differentiation behavior during those cultivation procedures has not yet been performed. Our study therefore aimed at answering the following question: is the redifferentiation pathway of chondrocytes altered by slight changes in the type of alginate biomaterial (pure alginate, alginate‐fibrin, alginate‐chitosan) and how do the cells behave in comparison to biomaterial‐free (pellet) three‐dimensional culturing? Monolayer‐expanded chondrocytes from healthy adult porcine knee joints were cultivated in alginate, alginate‐chitosan, alginate‐fibrin beads and as pellets up to 4 weeks. Quantitative PCR and Immunohistology were used to assess chondrogenic markers. Alginate‐fibrin—encapsulated chondrocytes behaved almost like monolayer chondrocytes. Alginate‐ and alginate‐chitosan encapsulation lead to a low chondrogenic marker gene expression. Although all 3D‐cultured chondrocytes showed a considerable amount of Sox9 expression, only pellet cultivation lead to a sufficient Collagen II expression. This puts the usage of alginate‐cultivated cartilage tissue engineering constructs under question. Fibrin addition is not beneficial for chondrogenic differentiation. Sox9 and Collagen II behave differently, depending upon the surrounding 3D‐environment. © 2009 American Institute of Chemical Engineers AIChE J, 2009     

Tuning structural durability of yeast-encapsulating alginate gel beads with interpenetrating networks for sustained bioethanol production

Microorganisms have become key components in many biotechnological processes to produce various chemicals and biofuels. The encapsulation of microbial cells in calcium cross-linked alginate gel beads has been extensively studied due to several advantages over using free cells. However, industrial use of alginate gel beads has been hampered by the low structural stability of the beads. In this study, we demonstrate that the incorporation of interpenetrating covalent cross-links in an ionically cross-linked alginate gel bead significantly enhances the bead's structural durability. The interpenetrating network (IPN) was prepared by first cross-linking alginate chemically modified with methacrylic groups, termed methacrylic alginate (MA), with calcium ions and subsequently conducting a photo cross-linking reaction. The resulting methacrylic alginate gel beads (IPN-MA) exhibited higher stiffness, ultimate strength and ultimate strain and also remained more stable in media either subjected to high shear or supplemented with chelating agents than calcium cross-linked alginate gel beads. Furthermore, yeast cells encapsulated in IPN-MA gel beads remained more metabolically active in ethanol production than those in calcium cross-linked alginate gel beads. Overall, the results of this study will be highly useful in designing encapsulation devices with improved structural durability for a broad array of prokaryotic and eukaryotic cells used in biochemical and industrial processes.