Some biological characteristics of hybridomas and parental myelomas cultivated in serum-free medium for long-term period

It has been developed in this laboratory that a serum-free medium, designated asDMI,is adaptive for long-term culture,freezing and resurgence in liquid nitrogen,and cloningof hybridoma and parental myelomas as well as for cell fusions.In this report,it is describedthat the myeloma cells grown in DMI for more than 3 months maintained their biological charac-teristics such as induction of aseites and subcutaneous somatic tumor in BALB/c mice and toler-ance toward 8-azaguanine,ete..However,the ability of the tumor cells to attach to glass wallwas decreased.The selecting assay of these cells in HAT medium(medium containing hypoxan-thine,aminopterin and thymidine)showed that the death time in DMI was similar to that in theconventional 15％ newborn calf serum-supplemented medium(RPS15).The hybridoma cellsadapted in DMI secrete monoclonal antibodies in quantities comparable to those produced inRPS15.

Cholesterol requirement for growth of rodent parental myeloma cells in serum-free medium

Cholesterol,a major lipid component of plasma membrane,is thoughtto have profound effects on the structure and function of cells.Mostanimal tis-sues are capable of synthesizing cholesterol de novo from acetate;however,thereare relatively few mammalian cells in vitro expressing an absolute requirement foran exogenous source of cholesterol.In this paper,it was shown that IR983F(983)rat myeloma cells and P3X63-AgS-U1(P3U1)and P3X63-Ag8.653(653)mousemy eloma cells which had been cultivated in serum-free medium for more than 6months required cholesterol in vitro for growth in serum-free medium.Optimalgrowth of P3U1,653 and 983 occurred in cholesterol concentration of 5,10 and15μg/ml,respectively.Moreover,it was demonstrated that the cholesterol couldbe replaced by human low density lipoprotein in a concentration of 10μg/ml butnot by mevalonic acid lactone.In contrast to the parental myeloma cells,hybridoma cells derived from the mouse myeloma cells which had been cultivatedin serum-free medium for more than 6 months did not require cholesterol.

Novel sericin-based hepatocyte serum-free medium and sericin's effect on hepatocyte transcriptome

AIM To develop a novel hepatocyte serum-free medium based on sericin,and to explore the effect of sericin on the hepatocyte transcriptome.METHODS A controlled trial comparing novel serum-free medium and other media: C3 A cells were cultured in our novel serum-free medium,Hepato ZYME,complete medium(DMEM/F12 with 100 m L/L FBS),and DMEM/F12,andthen cell attachment,proliferation,and function as well as the biocompatibility of the media were assessed.A comparative study of serum-free media with or without 2 mg/m L sericin: the effect of sericin on C3 A growth was assessed by cell viability and proliferation,the effect of sericin on C3 A cell cycle distribution was determined by flow cytometry,and the effect of sericin on the C3 A transcriptome was assessed by gene-chip array and RT-q PCR.RESULTS More C3 A cells attached to the plate containing our serum-free medium than to those containing Hepato ZYME and DMEM/F12 at 24 h post-seeding.Both the viability and proliferation rate of C3 A cells in sericin-based serum-free medium were superior to those of cells in Hepato ZYME and DMEM/F12(P < 0.001).The content of albumin and urea in our serum-free medium was significantly higher than that in Hepato ZYME and DMEM/F12 throughout the whole culture period(P < 0.001) and was similar to that in complete medium at day 3,4,and 5.In part 2,cell viability and proliferation were greater in the presence of 2 mg/m L sericin(P < 0.001),as was the proportion of cells in S phase(16.21% ± 0.98% vs 12.61% ± 0.90%,P < 0.01).Gene-chip array analysis indicated that the expression of CCR6,EGFR,and FOS were up-regulated by 2 mg/m L sericin,and RT-q PCR revealed that the expression of CCR6,EGFR,FOS,AKT1,JNK1,NFk B1,MMP-9,MEK2,ERK1/2 and MYC was upregulated by 2 mg/m L sericin(P < 0.05).CONCLUSION We developed a novel hepatocyte serum-free medium.Sericin probably enhances cell attachment through the CCR6-Akt-JNK-NF-κB pathway and promotes cell proliferation through CCR6-mediated activation of the ERK1/2-MAPK pathway.

A PRIMARY OBSERVATION OF TPA EFFECT ON SSV-NIH3T3 CELLS IN SERUM-FREE MEDIUM

The effect of TPA, a potent tumor promoter, on SSV-NIH3T3 cells in serum-free medium was investigated. TPA stimulated DNA synthesis of SSV-NIH3T3 cells on the third day of culture in SFM. In SDS-PAGF of medium conditioned by TPA-treated SSV-NIH3T3 cells (in SFM+TPA), the amounts of four proteins of 31.0 Kd, 28.5 Kd, 25.5 Kd and 13.5 Kd strikingly increased over that of non-TPA-treated counterpart (in SFM). The PDGF-like activity was also detected in CM of SFM+TPA. When insulin and EGF were drown off the SFM+TPA (SFM-Ins-EGF+TPA), TPA lost its ability to stimulate DNA synthesis of SSV-NIH3T3 cells on the third day and SDS-PAGE of the conditioned medium showed that the amounts of the four proteins noted above grately reduced. However, cells in SFM-Ins-EGF+TPA were in almost the same growth condition as cells in complete SFM+TPA on the third day of culture. Results were discussed in the paper.

Application of Serum-Free Culture Medium for Preparation of A-NK Cells

To compare the differences between proliferation and cytotoxicity of adherent natural killer （A-NK） cells cultured with serum-free medium AIMV and standard serum-containing medium in vitro, and also observe the assisting effect of IL-12 on the activation and the morphology character of IL-2-treated A-NK cells, cellular proliferation was evaluated by MTT method in vitro. The morphology of the target cells killed by A-NK cells was observed through electroscope. All of the A-NK cells cultured in serum-free medium AIMV could rapidly proliferate and keep high cytotoxicity compared with that in standard serum-containing medium. A-NK cells activated by both moderate-dose IL-2 and IL-12 were superior to the high-dose IL-2-treated A-NK cells. These data indicated that serum-free medium AIMV could replace standard serum-containing medium for culturing A-NK cells, and moderate-dose IL-2 and IL-12 could reduce side effects caused by high-dose IL-2. The study provided a new experimental basis for experimental and clinical preparation of A-NK cells. Cellular ＆ Molecular Immunology.

STUDY ON REPRODUCTIVE ENDOCRINOLOGY OF HUMAN PLACENTA--CULTURE OF HIGHLY PURIFIED CYTOTROPHOBLAST CELL IN SERUM-FREE HORMONE SUPPLEMENTED MEDIUM

A new method of long-term culture of cytotrophoblast cells in serum-free medium has been developed. Cytotrophoblast cells were isolated with cold trypsin and purified by unit gravity sedimentation through BSA density gradients. The cells were cultured in the FD medium with supplement of EGF, insulin, transferrin and sodium selenite. They could survive over three weeks. The results showed that both EGF and insulin stimulated hCG and progesterone secretion and that sodium selenite elevated hCG output but not progesterone secretion. Transferrin produced synergistic effect with EGF and insulin on hCG and progesterone secretion but it was ineffective when used alone. This study demonstrates that the four growth factors mentioned above are essential for the survival of cytotrophoblast cells in vitro. It is therefore suggested that EGF, insulin and selenium may possibly be involved in the regulation of hCG and progesterone secretion in the human placenta.

Enrichment of breast cancer stem cells using a keratinocyte serum-free medium

Background Keratinoyte serum-free medium （K-SFM） is a defined medium used to support the growth of primary keratinocytes and embryonic stem cell. The aim of this research was to optimize enrichment of breast cancer stem cells （CSCs） using K-SFM. Methods＇ A K-SFM was used to enrich CSCs from two breast cancer cell lines and a primary culture of breast cancer. RPMI-1640 supplemented with 10% fetal calf serum （FCS） was used as a control. CSCs were identified with flow cytometry using CD44＋/CD24-as molecular markers. The expression of a variety of CSC markers （Oct-4, ABCG2, Nanog, N-cadherin, and E-cadherin） was analyzed with real-time PCR. Results Much higher percentage of CSCs was achieved with K-SFM： 17.3% for MCF-7 cells, 17.4% for SKBR-3, and 20.0% for primary breast cancer culture. Less than 1% CSC was achieved using RPMI-1640 supplemented with 10% FCS. In comparison to the CSCs obtained with RPMI-1640, CSCs in the K-SFM expressed higher levels of Oct-4, ABCG2, Nanog and N-cadherin, and lower level of E-cadherin. Conclusion K-SFM is an optimal culture medium to maintain and to enrich breast CSCs.

A-NK细胞培养和活化方法的改进

目的:比较A-NK细胞在完全培养基(CM)和无血清培养基(AIMV)中体外扩增及杀伤肿瘤细胞作用,同时探讨IL-12对于A-NK/IL-2治疗的辅助作用及其杀伤的形态学观察。方法:用MTT法比较不同培养条件下的A-NK细胞体外增殖能力,并测定细胞体外杀伤肿瘤细胞的活性。通过做扫描和透射电镜,对A-NK细胞杀伤的肿瘤细胞进行形态学观察。结果:不同培养条件下的A-NK细胞均可在短期内大量扩增(P<0.05),且均有很强的杀伤肿瘤的活性(P<0.05)。被A-NK细胞杀伤的肿瘤细胞的死亡形式是溶解坏死(necrosis)和凋亡(apoptosis)。结论:AIMV可替代CM用于A-NK细胞的培养。联合应用IL-12和IL-2激活A-NK细胞优于单独使用IL-2,可减轻高剂量IL-2带来的副作用。此研究为A-NK细胞的实验室研究和将来临床应用推广奠定了新的实验基础。

Pilot-Scale Production of Lyophilized Inactivated Rabies Vaccine Candidate in Vero Cells under Fully Animal Component-Free Conditions Using Microcarrier Technology and Laboratory Animal Trials

The upstream process was carried out in an animal component-free medium on Cytodex 1 microcarriers. Recombinant trypsin is a non-animal derived protease used as an alternative to animal-derived trypsin. To inactivate recombinant trypsin, a soybean trypsin inhibitor (STI) should be added to the medium. A protocol was first tested in T-flasks and then passaged to 500 mL and 3 L spinner flasks. Cell detachment was completed in 10 - 12 min, and 0.4 g/L STI was added to a 3L spinner, and cells were transferred into a 30 L stirred tank bioreactor. On day 5, the cell density had reached its maximum (around 1.8 × 106 cells/mL). At an MOI of 0.3 with serum-free medium conditions, cell infection yielded a maximal rabies virus titer of 1.82 × 107 FFU/mL at 5 days. All cell culture conditions and virus growth kinetics in serum-free media were investigated. In conclusion, Vero cells were grown on Cytodex 1 with serum-free media and a high amount of rabies virus was obtained. A mouse challenge was used to determine the immune response to an inactivated rabies virus vaccine candidate. Also, we evaluated inactive rabies vaccine candidate safety, and immunogenicity in mice, sheep, horses, and cattle. We found that no horses, sheep, or cattle who were given vaccine IM at 3.2 IU/dose exhibited any clinical sign of disease and all developed high VNA titers (up to 10.03 IU/mL) by 3 - 4 WPI. After the accelerated stability studies, the lyophilized inactivated rabies vaccine candidate showed enough antigenic potency (2.6 IU/mL) in the mouse challenge test. Also, 18-month long-term stability studies showed enough immune response (1.93 IU/mL) on day 14. The activity of the vaccine candidate showed a good immune response and safety criteria that meet WHO requirements. This is the first pilot-scale mammalian cell-based viral rabies vaccine production study in Türkiye that used microcarriers.