Application of different microcarrier cell culture technologies in biological product field / 中国生物制品学杂志

@#The productivity of cells per unit area determines the scale-up potential of the cell culture process,and the largescale application of the microcarrier system provides space for the high-yield culture of anchorage-dependent animal cells. The microcarrier animal cell culture technology is suitable for efficient production process development and optimized amplification. In recent years,microcarrier-based culture technology has been widely used in various types of animal cell culture to produce many important biological products such as vaccines,enzymes,hormones,antibodies,interferons and other probiotics. In this paper,the research progress of domestic and foreign microcarrier cell culture technology,the comparison of new microcarriers and traditional microcarriers and their applications were reviewed,so as to provide reference for the in-depth research and application of large-scale cell culture technology based on new microcarriers.

Application of microcarrier technology in cartilage repairing: a review / 生物工程学报

Cartilage has poor self-recovery because of its characteristics of no blood vessels and high extracellular matrix. In clinical treatment, physical therapy or drug therapy is usually used for mild cartilage defects, and surgical treatment is needed for severe ones. In recent years, cartilage tissue engineering technology provides a new way for the treatment of cartilage defects. Compared with the traditional surgical treatment, cartilage tissue engineering technology has the advantages of small wound and good recovery. The application of microcarrier technology in the design of tissue engineering scaffolds further expands the function of scaffolds and promotes cartilage regeneration. This review summarized the main preparation methods and development of microcarrier technology in recent years. Subsequently, the properties and specific application scenarios of microcarriers with different materials and functions were introduced according to the materials and functions of microcarriers used in cartilage repair. Based on our research on osteochondral integrated layered scaffolds, we proposed an idea of optimizing the performance of layered scaffolds through microcarriers, which is expected to prepare bionic scaffolds that are more suitable for the structural characteristics of natural cartilage.

Tissue Regeneration of Human Mesenchymal Stem Cells on Porous Gelatin Micro-Carriers by Long-Term Dynamic In Vitro Culture

BACKGROUND: Tissue engineering is a multidisciplinary field which attracted much attention in recent years. One of the most important issue in tissue engineering is how to obtain high cell numbers and tissue regeneration while maintaining appropriate cellular characteristics in vitro for restoring damaged or dysfunctional body tissues and organs. These demands can be achieved by the use of three dimensional (3D) dynamic cultures of cells combined with cell-adhesive micro-carriers. METHODS: In this study, human mesenchymal stem cells (hMSCs) were cultured in a wave-bioreactor system for up to 100 days, after seeding on Cultisphere-S porous gelatin micro-carriers. Cell counting was performed at the time points of 7, 12, 17, 31 days and compared to those of hMSCs cultured under static condition. Higher growth and proliferation rates was achieved in wave-type dynamic culture, when cell culture continued to day 31. A scanning electron microscope (SEM) photographs, both live and dead and MTT assays were taken to confirm the survival and distribution of cells on porous gelatin micro-carrier surfaces. The results of histological stains such as hematoxylin and eosin, Masson’s trichrome, Alcian blue and Alizarin red S also showed improved proliferation and tissue regeneration of hMSCs on porous gelatin micro-carriers. CONCLUSION: The experimental results demonstrated the effect and importance of both micro-carriers and bioreactor in hMSC expansion on cell proliferation and migration as well as extracellular matrix formation on the superficial and pore surfaces of the porous gelatin micro-carriers, and then their inter-connections, leading to tissue regeneration.

Establishment of human gastric cancer model in normal immune mice based on micro-carrier 6 / 中国肿瘤临床

Objective:To establish a mouse model of gastric cancer by inoculating MKN45 cells into mice with normal immune function utilizing microcarrier technology. Methods:A total of 60 male C57BL/6 mice were randomly divided into three groups, namely, 2D, con-trol, and 3D groups, according to the coculture system of MKN45 and microcarrier. The mouse models of gastric carcinoma were estab-lished by hypodermic injection. The time of tumorigenesis, rate of tumor formation, and pathological features were observed in each group. Results:In the 3D group, the time of tumor formation was short, whereas the rate of tumor formation was high (80%). No de-tectable tumor formations were observed in the 2D and control groups. HE and immunohistochemical staining of the transplantation tumor model showed evident characteristics of human gastric cancer. Conclusion:A human gastric cancer model in normal immune mice was successfully established. The onset and development mechanism of gastric cancer could be more effectively investigated in mice with normal immune function through this model. Moreover, a more valuable and new animal model for the research and devel-opment of anticancer drug was established.

Study of nanofiber microcarriesr/collagen gel on in vitro cultivation of hepatocytes / 国际生物医学工程杂志

Objective To investigate cell's viability and functions when human hepatocyte cell HepG2 and microcarriers were embedded in collagen gel and cultured in vitro.Methods Ultrasound concussion crushing method was used to get nanofiber microcarrier.HepG2 and microcarriers were embedded in collagen gel and cultured in 12 d.The albumin (ALB),urea and lactate dehydrogenase (LDH) levels in the nutrient solution were measured.Results The cells in nanofiber nanofiber microcarrier/collagen gel group were gathered around the microcarrier and formatted aggregates,the ALB and urea levels increased steadily and reached maximum in day 9,then decreased; In collagen gel without nanofiber microcarrier group cells uniformly distributed,the ALB and urea levels reached maximum in day 3,then decreased rapidly,and on day 6 majority of cells dead.Conclusion Gel entrapped nanofiber microcarriers and hepatoeytes is a high-density and longtime culture method which can possibly be used in the bioartificial liver system.

Evaluating kinetic and physiological features of rCHO-K1 cells cultured on microcarriers for production of a recombinant metalloprotease/disintegrin

We present kinetic and physiological data regarding the culturing of rCHO-K1 cells on various microcarriers, to evaluate the potential of this culture strategy for mass production of these cells and expression of a recombinant disintegrin. Cultures were performed in 500 mL spinner flasks in DMEM culture medium with 10 percent v/v fetal calf serum, gently shaken at 37 C, pH 7.4, in a 10 percent v/v CO2 atmosphere. The following values were obtained, respectively, for the adhesion time-constant Ka (h) and specific growth rate micron max (d-1) on each microcarrier: Cytodex 1 (0.91, 0.45), Cultispher S (0.28, 0.34), Immobasil FS (0.85, 0.52) and Pronectin F (5.12, 0.67). Metabolic characteristics showed some variation among the cultures with the four microcarriers, the most significant being the higher production of ammonia with microcarriers coated with adhesive molecules (Cultispher S and Pronectin F) relative to the uncoated carriers (Cytodex 1 and Immobasil FS). Experiments where the DMEM medium was gradually replaced by the serum-free medium (CHO-SFM-II) revealed important advantages over media containing serum, not only for assay purposes, but also for purification of the disintegrin. Altogether these results demonstrate that cultures on microcarriers, especially on Pronectin F, show good potential for larger scale cultures of rCHO-K1 cell.

Optimal Number of Hepatocytes per Microcarrier in Spheroid Culture using Cytodex 3 Microcarrier

PURPOSE: The mass cultivation of functional hepatocytes is a key factor of a bioartificial liver. Combining spheroid and microcarrier cultures has been applied for enhancing the cell viability and metabolic activities. Hence, the optimal number of hepatocytes per microcarrier was investigated. METHODS: Firstly, spheroid cultures were carried out with 1 g Cytodex 3 microcarrier plus 2 x 10(9), 4 x 10(8) and 8 x 10(7) viable hepatocytes per flask. The numbers of hepatocytes per microcarrier were approximately 666.7, 133.3 and 26.7, respectively. The control group consisted of a spheroid culture of 4 x 10(8) hepatocytes without any microcarrier. According to the primary experimental results, spheroid cultures with 1 x 10(8) of hepatocytes plus 1 g, 2 g and 3 g of the Cytodex 3 microcarrier were performed. The numbers of hepatocytes per microcarrier were approximately 33.3, 16.7 and 11.1, respectively. The control group consisted of a spheroid culture of 1 x 10(8) hepatocytes. The cell viabilities were assayed using a Cell Counting Kit-8; with the albumin production assayed using ELISA. RESULTS: According to the primary experiment, the group consisting of 26.7 hepatocytes per microcarrier showed the highest viability (P<0.01). However, there was no statistical difference in the albumin production between the groups (P=0.744). The second Experiment showed the groups consisting of 11.1 and 16.7 hepatocytes per microcarrier had higher viabilities than the other hepatocyte and control groups (P<0.01). The albumin production was similar for each group (P=0.187). CONCLUSION: With respect to their application to a bioartificial liver, about 130 hepatocytes per microcarrier was appeared to be good for the mass cultivation of a hepatocytes spheroid culture using the Cytodex 3 microcarrier.

Micromechanism and biocompatibility of cartilage derived microcarrier for injectable tissue engineering cartilage / 中国矫形外科杂志

[Objective]To improve the preparative method of catilage derived microcarrier and evaluate the micromechanism and biocompatibility of this metarial for injectable tissue engineering cartilage.[Methods]Fresh porcine articular cartilage were obtained and shattered in the iso-osmia liquid in 4℃.After gradient centrifugation,150-300 ?m size cartilage micelles were gathered and were subjected to 1% Triton X-100 once and physiological saline twicely.The structure of specimens were observed and assessed by inverted phase contrast microscopy,environmental scanning electron microscope.And the composition of these specimens were stained with haematoxylin-eosin,safranin-O,toluidine blue and immunohistochemistry of collagen type Ⅱ.The microcarriers were seeded with rabbit bone mesenchymal stem cells(BMSCs) and cocultured with Rotary Cell Culture System(Rotary Cell Culture System,RCCS TM).[Results]The cartilage particles had fiocculus apparence.There were numerous villus on the surface of these cartilage micelles,which were stained positive with the immunohistochemistry of collagen type Ⅱ.The inner part of these cartilage micelles were stained positively with safranin-O and toluidine blue.After coated with BMSCs and cultured in the RCCSTM,the cells grew well on the surface of the cartilage micelles and the latter could be disperse again after blowed with pipette.[Conclusion]The cartilage micelles,which have large surface area and good boicompatibility,is a new kind of microcarrier for injectable tissue engineering cartilage.

A quick way in vitro for amplification of goat chondrocytes by RCSS / 中国矫形外科杂志

[Objective]To establish a method for obtaining well-differentiated goat chondrocytes quickly in large scale by RCSS using microcarriers technique. [Method]Articular chondrocytes were harvested from goat by sequential digestion with trypsin and collagenase , and then grown in RCSS bioreactor culture system which contained the Cytodex-3 microcarriers in the culture medium (DMEM) . Growth of chondrocytes on Cytodex-3 microcarriers was observed dynamically under phase contrast microscope . Immunocytochemical analysis was performed for type Ⅰcollagen and type Ⅱcollagen. [Result]The articular chondrocytes attached rapidly to the surface of Cytodex-3 microcarriers. Quick growth of these cells was observed after they fully spread onto the microcarriers. The density of chondrocytes increased by 15～17 times in later stage of culture as compared with the initial density. The harvested chondrocytes had no detectable staining for collagen type Ⅰ, but stained intensively for collagen type Ⅱ. [Conclusion]Microcarrier culture of chondrocytes can yield a large quantity of goat cells within a short time ,which will be of benefit for banking cartilage cells for reconstruction of impaired cartilage by way of tissue engineering.

Construction of three dimensional in vitro culture model of pulmonary adenocarcinoma and detection of its drug resistance / 第三军医大学学报

Objective To explore an optimal model of three dimensional in vitro cell culture for simulating solid tumors in vivo . Methods The model of three dimensional cell culture was constructed under the conditions of inhibiting the cell wall attachment and stirring the medium. Multicellular spheroids (MCS) were cultured using microcarriers (CutiSpher). Drug sensitivity of monolayer cells (MC) and MCS was tested by MTT staining and cytometry, respectively. Ultrastructures of the MC and MCS were observed by transmission electron microscopy. Results Cells in three dimensional cell culture model without microcarriers were compacted into mass at 4 d while cells in MCS were found to attach to the microcarriers at 0.5 h. MCS had more than two layers of cells growing within it at 5 d. Compared with MC, MCS was more resistant to the anticancer drug, and had more plenty of organell and microvilli with more extensive and compact cell adhesion. Conclusion MCS has strong developmental properties and can simulate the cell cell interactions in vivo , especially cell adhesion, which may contribute to the drug resistance of MCS.

In vitro amplification of immortalized chondrocytes with micro-carriers in RCCS / 第三军医大学学报

Objective To explore the method and technique to gain adequate seed cells for cartilage tissue engineering Methods hTERT gene was introduced into rabbit mandibular condylar chondrocytes by eukaryotic vector Rapidly proliferated immortalized chondrocytes in positive clones in micro carrier rotary cell culture system (RCCS) were screened and selected The growth of immortalized chondrocytes and the metabolic rate were observed Collagen type Ⅱ expression of immortalized chondrocytes of the experimental groups was observed The immunohistochemical results were compared with those of the control groups Results The immortalized chondrocytes in experimental groups could grow rapidly with a high metabolic rate and shorter population doubling time (PD) ( P

In vitro amplification of immortalized keratinocytes with micro-carriers in rotary cell culture system / 第三军医大学学报

Objective To explore the way and technique to gain adequate seed cells for skin tissue engineering sufficiently. Methods Human telomerase reverse transcriptase (hTERT) gene was introduced into rabbit keratinocytes by eukaryotic vector. The positive clones were selected and cultured in microcarrier-RCCS. The growth of immortalized keratinocytes was observed, and the metabolic rate and pan-cytokeratins (AE1/AE3) expression of immortalized keratinocytes in experimental groups were detected and compared with those in the control group. Results The immortalized keratinocytes in the experimental groups grew rapidly and had a high metabolic rate and shorter population doubling time (PD) (P

The Effect of 3-Dimensional Microcarrier Culture on Chondrocyte Proliferation and Matrix Production / 대한정형외과연구학회지

PURPOSE: Injury to articular cartilage is likely progressive to develop arthritis and dysfunction of joint because its regeneration is limited. Transplantation of cultured autologous chondrocytes was proved to be a satisfactory treatment method and could prevent a dysfunction of joint. However chondrocytes propagated in monolayer culture lose their original characteristics by assuming a morphologic shift to fibroblast like cell and biosynthetic shift to production of collagen type I from type II and to decrease in synthesis of DNA and proteoglycans. A three dimensional culture of articular chondrocytes using microcarrier was performed to overcome disadvantages of phenotypic changes in monolayer culture. MATERIALS & METHODS: Cultured articular chondrocytes, 6 times passed in 13 weeks, were innoculated and propagated in flasks for monolayer culture and spinner flasks for three dimensional culture respectively for 2 weeks. RESULTS: Cells in three dimensional culture proliferated faster and showed more markedly increase in cell number than those in monolayer culture. Expression of type II collagen was observed in three dimensional culture, but was hardly observed in monolayer culture. CONCLUSION: Degree of synthesis of DNA and proteoglycans also more markedly increase in three dimensional culture than in monolayer culture. Our experiments demonstrate that three dimensional culture of articular chondrocyte using microcarrier provides increase in growth and reexpression of phenotype of chondrocytes.

The Effect of 3-Dimensional Microcarrier Culture on Chondrocyte Proliferation and Matrix Production / 대한정형외과연구학회지

PURPOSE: Injury to articular cartilage is likely progressive to develop arthritis and dysfunction of joint because its regeneration is limited. Transplantation of cultured autologous chondrocytes was proved to be a satisfactory treatment method and could prevent a dysfunction of joint. However chondrocytes propagated in monolayer culture lose their original characteristics by assuming a morphologic shift to fibroblast like cell and biosynthetic shift to production of collagen type I from type II and to decrease in synthesis of DNA and proteoglycans. A three dimensional culture of articular chondrocytes using microcarrier was performed to overcome disadvantages of phenotypic changes in monolayer culture. MATERIALS & METHODS: Cultured articular chondrocytes, 6 times passed in 13 weeks, were innoculated and propagated in flasks for monolayer culture and spinner flasks for three dimensional culture respectively for 2 weeks. RESULTS: Cells in three dimensional culture proliferated faster and showed more markedly increase in cell number than those in monolayer culture. Expression of type II collagen was observed in three dimensional culture, but was hardly observed in monolayer culture. CONCLUSION: Degree of synthesis of DNA and proteoglycans also more markedly increase in three dimensional culture than in monolayer culture. Our experiments demonstrate that three dimensional culture of articular chondrocyte using microcarrier provides increase in growth and reexpression of phenotype of chondrocytes.