Effect of bioreactor scale-up culture of micro-carrier Vero cells after extra-tank trypsinization on virus-producing ability of rabies virus CTN-1Ⅴ strain / 中国生物制品学杂志

@#Objective To investigate the effect of amplification culture of micro-carrier Vero cells from 30 L bioreactor to 300 L bioreactor after extra-tank trypsinization on the virus-producing ability of rabies virus(RABV)CTN-1Ⅴ strain.Methods The 140-passage of Vero cells were cultured at 37 ℃ for 72-120 h,then amplified by passaging at a cell density ratio of 1∶4 into the 10 × cell factory. After incubation at 37 ℃ for 72-120 h,the monolayer cells were detached and inoculated into the 30 L bioreactor with micro-carriers 7-10 g/L,culture temperature 37 ℃,pH 7. 0-7. 4,dissolved oxygen 30%-80%,stirring speed 10-50 r/min,and continuous perfusion culture 72-120 h. Total three batches of microcarrier Vero cells were cultured,which were amplified to the 300 L bioreactor after extra-tank trypsinization,with microcarrier5-8 g/L,culture temperature 37 ℃,pH 7. 0-7. 4,dissolved oxygen 30%-80%,stirring speed 30-80 r/min,and perfusion culture 72-120 h. RABV CTN-1Ⅴ was inoculated at the MOI of 0. 05,and the virus solution was harvested every 24 h and detected for the virus titer and antigen content. Results The cell density was about 1 × 10~7 cells/mL after culture for 96 h in the 30 L bioreactor,and was about 7. 4 × 10~6 cells/mL after culture for 96 h in the 300 L bioreactor. At 96 h after virus inoculation,the virus harvest solution reached the peak potency,with the average virus titer of 6. 8 lgLD_(50)/mL and the average antigen content of 2. 58 IU/mL. Conclusion The scale-up culture process of micro-carrier Vero cells after extra-tank trypsinization from 30 L bioreactor to 300 L bioreactor is stable and feasible,with no significant effect on the virus-producing ability of RABV CTN-1Ⅴ strain,which provides a reference for the large-scale production of inactivated RABV vaccine

Melanocyte spheroids are formed by repetitive long-term trypsinization

Background: Autologous melanocyte transplantation plays an important role in the treatment of vitiligo. Objective: Previous studies have indicated that, compared with melanocytes growing in monolayers, melanocyte spheroids have a better survival in growth factor- and serum-deprived conditions. Methods: Melanocyte spheroids were obtained from human epidermis by repetitive long-term trypsinization and maintained an aggregated morphology for a short period in certain conditions. Results: Melanocyte spheroids were capable of growing into normal dendritic melanocytes in monolayer when they were harvested and reinoculated in 24-well plates. Immunohistochemical analysis of the melanocyte spheroids revealed that they were positive for HMB45, a melanosome-specific marker. No melanomas occurred when melanocyte spheroids were transplanted into mice. Conclusion: Our study provides a promising approach for melanocyte transplantation to treat vitiligo.

Neutral red staining and trypsin treatment to study viability and culture yield of Dermatophytes.

Dermatophytosis is common fungal infection of human being. To diagnose dermatophytic infections microscopic examination should be followed by culture which is essential step. Many times fungus may fail to grow on culture even after direct microscopy is positive due to non-Viability of fungus and it has been revealed that trypsinization enhances the rate of isolation of fungus on culture. Therefore, the study was undertaken with an aim to look for the viability and yield of dermatophytes on neutral red staining and trypsinization respectively.

Non-cultured epidermal suspension in vitiligo: From laboratory to clinic.

Background: Medical treatments are ineffective in many patients and surgical methods have therefore been developed. Objective: A review of autologous non-cultured melanocyte grafting techniques is proposed to obtain a successful repigmentation of vitiligo macules. Methods: Initially in 1992, we had developed a simplified grafting method which was carried out in the following two steps: production of blisters on the depigmented lesions by freezing with liquid nitrogen and injection in each blister of a non-cultured suspension of epidermal cells. The cellular suspension was obtained from samples of skin of the hair scalp after trypsinization. This very simple technique could be used at the dermatologist's clinic. Since 1998 (Olsson MJ, Juhlin L), quite comparable but improved and more sophisticated techniques have been proposed for the surgical treatment of vitiligo. These techniques require a laboratory set up to perform the melanocyte transplantation. The donor zone was usually taken on the gluteal region. The time of trypsinization was reduced to 60 minutes at 37΀C and the centrifuged cellular suspension added with hyaluronic acid (Van Geel) was directly applied on a dermabraded or laser abraded vitiligo lesions. Results: Whatever the technique chosen, repigmentation was evident within 25 to 30 days. Coalescence of the pigmented areas was spontaneously observed or obtained after UVB radiation. It is obvious that the complete repigmentation occurred more rapidly with the recent techniques compared with the initial method, but the efficiency was quite similar. Conclusion: The use of non-cultured epidermal suspension appears to be an effective, safe, and simple method for treating patients with achromic areas lacking melanocytes.

Optimization of primary culture of neural stem cells / 中华神经医学杂志

Objective To study the influence of digestion times of low concentration trypsin on the proliferation and apoptosis of neural stem cells (NSCs) in the hippocampus of neonate rats.Methods Hippocampus of neonatal rats (within 24 h) were taken out, and treated with trypsin at 1.25g/L concentration and 37 ℃ for 5, 10, 15, 20 and 25 minutes; unicellular suspension was then successfully got and primary culture and subculture were performed. Effects of trypsinization on cell viability and growth of NSCs were compared by observing the cell morphology and Trypan blue staining.The 5-bromodeoxyuridine labeling was performed to assess the self-renewing and proliferative activities of NSCs. Fluorescence immunocytochemistry was carried out to examine the expressions of BrdU and nestin. Apoptosis was measured by Annexin V-FITC/PI assay and flow cytometry. Results Primary and passage culture of NSCs enjoyed rapid proliferation and formation of neurospheres. The neurosphere cells expressed NSCs specific marker nestin by immunofluorescence; all the neurosphere cells could incorporate BrdU into the nucleus; of the neurospheres obtained from the 3rd, 5th and 7th d, those digested for 15 rain enjoyed the highest level of NSCs neurospheres, the highest BrdU labeled clone and the lowest cell apoptosis as compared with those digested for 5, 10, 20 and 25 min (P<0.05). Conclusion The NSCs isolated from the hippocampus of neonatal rats have the ability of proliferation in vitro. And 1.25 g/L concentration of trypsin with digestion times could positively change the proliferative and apoptosis capacity of NSCs: too short or long digestion times can inhibit the proliferation of NSCs and induce the apoptosis of NSCs; the longer the digestion time, the higher the apoptosis of NSCs.