GMP compliant isolation of mucosal epithelial cells and fibroblasts from biopsy samples for clinical tissue engineering

Engineered epithelial cell sheets for clinical replacement of non-functional upper aerodigestive tract mucosa are regulated as medicinal products and should be manufactured to the standards of good manufacturing practice (GMP). The current gold standard for growth of epithelial cells for research utilises growth arrested murine 3T3 J2 feeder layers, which are not available for use as a GMP compliant raw material. Using porcine mucosal tissue, we demonstrate a new method for obtaining and growing non-keratinised squamous epithelial cells and fibroblast cells from a single biopsy, replacing the 3T3 J2 with a growth arrested primary fibroblast feeder layer and using pooled Human Platelet lysate (HPL) as the media serum supplement to replace foetal bovine serum (FBS). The initial isolation of the cells was semi-automated using an Octodissociator and the resultant cell suspension cryopreservation for future use. When compared to the gold standard of 3T3 J2 and FBS containing medium there was no reduction in growth, viability, stem cell population or ability to differentiate to mature epithelial cells. Furthermore, this method was replicated with Human buccal tissue, providing cells of sufficient quality and number to create a tissue engineered sheet.

Tissue Specific Differentiation of Human Chondrocytes Depends on Cell Microenvironment and Serum Selection

Therapeutic options to cure osteoarthritis (OA) are not yet available, although cell-based therapies for the treatment of traumatic defects of cartilage have already been developed using, e.g., articular chondrocytes. In order to adapt cell-based therapies to treat OA, appropriate cell culture conditions are necessary. Chondrocytes require a 3-dimensional (3D) environment for redifferentiation after 2-dimensional (2D) expansion. Fetal bovine serum (FBS) is commonly used as a medium supplement, although the usage of a xenogeneic serum could mask the intrinsic behavior of human cells in vitro. The aim of this study was to compare human articular chondrocytes cultivated as monolayers (2D) and the development of microtissues (3D) in the presence of FBS with those cultivated with human serum (HS). Evaluation of the expression of various markers via immunocytochemistry on monolayer cells revealed a higher dedifferentiation degree of chondrocytes cultivated with HS. Scaffold-free microtissues were generated using the agar overlay technique, and their differentiation level was evaluated via histochemistry and immunohistochemistry. Microtissues cultivated in the medium with FBS showed a higher redifferentiation level. This was evidenced by bigger microtissues and a more cartilage-like composition of the matrix with not any/less positivity for cartilage-specific markers in HS versus moderate-to-high positivity in FBS-cultured microtissues. The present study showed that the differentiation degree of chondrocytes depends both on the microenvironment of the cells and the serum type with FBS achieving the best results. However, HS should be preferred for the engineering of cartilage-like microtissues, as it rather enables a "human-based" situation in vitro. Hence, cultivation conditions might be further optimized to gain an even more adequate and donor-independent redifferentiation of chondrocytes in microtissues, e.g., designing a suitable chemically-defined serum supplement.

Isotyping antibodies produced from hybridoma A6G11C9

Hybridoma technology was discovered in 1975 to produce the monoclonal antibodies. By this way, we can produce a desired antibody in large amounts. From a single hybrid cell line, they grow and develop to produce a monoclonal antibody with large enough quantities to use for the research, treatment and diagnosis. The level of the antibody producing is depended on the cell density and the incubation period. The growth capacity of each hybrid cell line depends on the composition of the substances in the culture medium of animal cells. Among them, fetal bovine serum is the most commonly used serum-supplement for the in vitro cell culture. This is due to it having a very low level of antibodies and containing more growth factors. In the previous studies, the hybrid cell line (designed A6G11C9) was the best one secreting the highest anti-A monoclonal antibodies, whose specificity agglutinaned human red blood cells containing A antigen. This report showed the result of the study on the coditional culture and isotyping of the immunoglobulin. The hybrid cell line A6G11C9 was cultured in the DMEM medium with different level of fetal bovine serum. As the results, this hybridoma line grow in the DMEM medium containing 10% fetal bovine serum is five times faster than in the DMEM medium with 1% fetal bovine serum. The maximum of the cell density are 11.106 cells/ml after 50 hours innoculation. The maximum of the titer from culture supernatant are 1/1024 after 100 hours innoculation. The monoclonal antibodies derived from hybrid cell line A6G11C9 are IgM heavy chain and the kappa light chain.

Effect of Fetal Calf Serum and Mid Estrus Cow Serum on In Vitro Maturation and Fertilization of Oocytes from Crossbred Cattle in the Tropics

The objective of the study was to examine the effect of fetal calf serum (FCS) and mid estrus cow serum (ECS) in culture media on in vitro maturation (IVM) and in vitro fertilization (IVF) of oocytes from crossbred cattle in the tropics. Oocytes from abattoir ovaries were cultured in three different maturation media at 39oC temperature, 5% CO2 tension with maximum humidity for 24 h. TCM-199 containing 25mM HEPES, 1mM glutamine L, 2.2mg/mL sodium bicarbonate, antibiotics, 22 µg/mL pyruvate, 1µg/mL estradiol-17β, 0.5µg/mL FSH and 0.06 IU hCG without serum supplement used as treatment-1 and the above media were further supplemented with 10% FCS as treatment-2. In treatment-3, instead of 10% FCS, 20% heat inactivated ECS (serum collected in mid estrum) was used as serum supplement. Oocytes with maximum degree of cumulus expansion were selected as matured and used for further IVF studies using frozen semen. The IVF medium consisted of Fert-TALP medium supplemented with 1µM epinephrine, 10µM hypotaurine, 20µM pencillamine and 0.56µg/ml heparin. Culture conditions set for IVF were 39oC temperature, 5% CO2 tension with maximum humidity. Oocytes showing sperm penetration evidence like presence of enlarged sperm head, male pronuclei with its accompanying sperm tail in the cytoplasm, oocytes with two pronuclei and a clear second polar body but without a sperm tail were considered as fertilized. Significantly higher result of cumulus expansion percentage (p< 0.05) was observed when oocytes matured in media supplemented with FCS as compared to other two treatments. There was no influence of source of serum in maturation media on further IVF of matured oocytes in this study. Int J Appl Sci Biotechnol, Vol 4(3): 308-310

81 VITRIFICATION OF IMMATURE PORCINE CUMULUS - OOCYTE COMPLEXES IN A CHEMICALLY DEFINED SOLUTION

A high concentration of serum supplements has been contained in basic vitrification solutions to protect plasma membranes. The objective of this study was to examine if vitrification of immature porcine oocytes could be achieved successfully in a chemical-defined solution containing 0.6 mg mL–1 hydroxypropyl cellulose (HPC). Cumulus–oocyte complexes (COC) were aspirated from follicles 3 to 6 mm in diameter in abattoir-derived porcine ovaries. The COC or denuded oocytes were vitrified according to a commercial protocol of Cryotop (Kitazato BioPharma Co. Ltd., Shizuoka, Japan) with original solutions or modified ones that had serum supplement replaced with HPC. After vitrification and warming, viabilities of oocytes and cumulus cells were evaluated under a fluorescent microscope after staining with fluorescein diacetate and propidium iodide (Table 1). Statistical analyses of results from 4 replicated trials were performed by ANOVA with a Bonferroni/Dunn post hoc test (significance, P < 0.05). Although viabilities of vitrified/warmed oocytes in all groups were significantly lower (82.7–89.1%) than those of fresh controls (99.5%), there were no significant differences among vitrified groups. When COC were vitrified, viability of cumulus cells (54.3%) in HPC group was not different from that of nonvitrified controls (72.5%) but higher than that in the original solution group (48.1%). In conclusion, these results demonstrate that HPC rather than serum supplement could be a suitable chemically defined supplement for vitrification of immature porcine COC. Table 1.Viabilities of vitrified/warmed porcine oocytes and cumulus cells1