scholarly journals Patterning of diverse mammalian cell types in serum free medium with photoablation

2009 ◽  
Vol 25 (2) ◽  
pp. 594-603 ◽  
Author(s):  
Vipra Dhir ◽  
Anupama Natarajan ◽  
Maria Stancescu ◽  
Anindarupa Chunder ◽  
Neelima Bhargava ◽  
...  
Keyword(s):  
Mammalian Cell ◽  
Cell Types ◽  
Serum Free Medium ◽  
Free Medium ◽  
Serum Free

High-Level Expression of Porcine Factor VIII from Murine Mesenchymal Stem Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 5281-5281
Author(s):  
Bagirath Gangadharan ◽  
H. Trent Spencer ◽  
Ernest T. Parker ◽  
Christopher B. Doering
Keyword(s):  
Gene Transfer ◽  
Ex Vivo ◽  
Cell Types ◽  
Mrna Levels ◽  
Serum Free Medium ◽  
Free Medium ◽  
High Level Expression ◽  
Serum Free ◽  
Fviii Activity ◽  
High Level

Abstract Low-level expression of human factor VIII (fVIII) has limited the success of clinical trials for hemophilia A using both ex vivo and in vivo gene transfer methods. Ex vivo genetic modification provides increased control of gene transfer and permits thorough characterization of transgene copy number, chromosomal integration site(s), and expression levels prior to transplantation. A subpopulation of adherent bone marrow-derived cell types, termed mesenchymal stem cells (MSCs), comprise an attractive target cell type for ex vivo gene transfer due to their accessibility, ability to differentiate into multiple cell types, and long-term survival following transplantation. Recently we demonstrated, in vitro, that recombinant B-domain-deleted porcine fVIII (rp-fVIII) is expressed at levels 10 – 14-fold greater than recombinant B-domain-deleted human fVIII (rh-fVIII) due to an enhanced rate of secretion from baby hamster kidney-derived (BHK-M) cells. Additionally we found that only the A1 and activation peptide-A3 domain sequences of porcine fVIII are necessary to retain high-level expression of hybrid human/porcine fVIII constructs. Here we report the expression of rp-fVIII from murine MSCs isolated from exon-16 fVIII knockout mice following ex vivo retroviral transduction. MSCs were transduced with ecotropic envelope-pseudotyped murine stem cell virus containing a rp-fVIIII transgene at an multiplicity of infection of 2 – 5 functional viral particles/target MSC. Following this transduction regimen the cell population harbored an average of 1.8 proviral genomes/cell as determined by quantitative real-time PCR. During culture in growth medium supplemented with 20% fetal bovine serum (FBS) rp-fVIII-transduced MSCs demonstrated a steady-state level of 2,400 fVIII mRNA transcripts/cell and an apparent fVIII production rate of 174 units/106 cells/24 hr as determined by quantitative real-time RT-PCR and one-stage clotting assay, respectively. However, when cultured in serum-free medium the mRNA levels decreased to 950 transcripts/cell and apparent fVIII production was reduced to 14 units/106 cells/24 hr. The latter mRNA/fVIII expression ratio is in agreement with that previously reported from stably transduced BHK-M clonal cell lines. In contrast the former mRNA levels are lower than predicted from the observed fVIII activity levels. The activation quotient (ratio of apparent fVIII activity following thrombin pre-treatment to non-thrombin treated baseline fVIII activity) increased 17-fold when the cells were cultured in serum-free versus serum-containing medium. Additionally, SDS-PAGE analysis of immunoprecipitated fVIII protein from serum-containing and serum-free medium revealed the presence of activated fVIII (fVIIIa) in conditioned medium samples containing FBS. Highly purified rp-fVIII from transduced-MSCs cultured in serum-free medium displayed similar relative mobility to BHK-M produced rp-fVIII upon SDS-PAGE analysis. These data warn against the determination of fVIII activity in serum-containing medium using the one-stage coagulation assay due to the presence of activated fVIII with high specific activity. Based on these results it is reasonable to predict that hemophilia A could be cured using high-level expression fVIII constructs by transplantation of a feasible number (106 - 108) of cells containing a single copy of rp-fVIII or other high-level expression hybrid human/porcine fVIII transgenes.

scholarly journals Human Thymic Epithelial Cells in Serum-Free Culture: Nature and Effects on Thymocyte Cell Lines

1992 ◽  
Vol 2 (2) ◽  
pp. 111-121 ◽  
Author(s):  
Carsten Ropke ◽  
Jette Elbroend
Keyword(s):  
Monoclonal Antibodies ◽  
Epithelial Cells ◽  
Cell Types ◽  
Thymic Epithelial Cells ◽  
Serum Free Medium ◽  
Free Medium ◽  
Serum Factors ◽  
Serum Free ◽  
Functional Activities ◽  
Thymocyte Proliferation

Thymic epithelial cells (TEC) have been cultured for several months and/or for 4 to 5 transfers in a growth factor-defined serum-free medium without concurrent growth of other cell types. The use of monoclonal antibodies andαMAM-6 indicated that the majority of TEC were of medullary origin. The vast majority of cells were positive for LFA-3 and class I, and class II expression, was low or absent. Supernatants from the cultures were shown to contain IL-1ß, IL-6, and M-CSF. Coculture of cloned subpopulations of thymocytes and TEC showed effects of TEC and of secreted ILs on thymocyte proliferation. High percentages of TEC were able to bind DN, DP, or SP thymocyte populations, partly via CD2-LFA-3 adhesion. Thus, it is possible to culture TEC without unknown serum factors and with maintenance of functional activities.

Exogeneous factors are not necessary in attachment, spreading and polarization of hela cells

1978 ◽  
Vol 36 (2) ◽  
pp. 310-311
Author(s):  
W. Liebrich
Keyword(s):  
Hela Cells ◽  
Calf Serum ◽  
Glass Tube ◽  
Serum Free Medium ◽  
Nacl Solution ◽  
Free Medium ◽  
Minimum Essential Medium ◽  
Serum Free ◽  
All Solutions ◽  
Glass Support

HeLa cells were grown for 2-3 days in EAGLE'S minimum essential medium with 10% calf serum (S-MEM; Seromed, München) and then incubated for 24 hours in serum free medium (MEM). After detaching the cells with a solution of 0. 14 % EDTA and 0. 07 % trypsin (Difco, 1 : 250) they were suspended in various solutions (S-MEM = control, MEM, buffered salt solutions with or without Me++ions, 0. 9 % NaCl solution) and allowed to settle on glass tube slips (Leighton-tubes). After 5, 10, 15, 20, 25, 30, 1 45, 60 minutes 2, 3, 4, 5 hours cells were prepared for scanning electron microscopy as described by Paweletz and Schroeter. The preparations were examined in a Jeol SEM (JSM-U3) at 25 KV without tilting.The suspended spherical HeLa cells are able to adhere to the glass support in all solutions. The rate of attachment, however, is faster in solutions without serum than in the control. The latter is in agreement with the findings of other authors.

Hemoglobin Enhances the Binding of Bacterial Endotoxin to Human Endothelial Cells

1996 ◽  
Vol 76 (02) ◽  
pp. 258-262 ◽  
Author(s):  
Robert I Roth
Keyword(s):  
Endothelial Cells ◽  
Binding Protein ◽  
Bacterial Endotoxin ◽  
Dependent Manner ◽  
Serum Free Medium ◽  
Free Medium ◽  
Human Endothelial Cells ◽  
Serum Factors ◽  
Serum Free ◽  
Lps Binding

SummaryHuman endothelial cells, when incubated with bacterial endotoxin (lipopolysaccharide, LPS), modify their surface in association with prominent production of procoagulant tissue factor (TF) activity. This deleterious biological effect of LPS has been shown previously to be enhanced approximately 10-fold by the presence of hemoglobin (Hb), a recently recognized LPS binding protein that causes disaggregation of LPS and increases the biological activity of LPS in a number of in vitro assays. The present study was performed to test the hypothesis that Hb enhances the LPS-induced procoagulant activity of human umbilical vein endothelial cells (HUVEC) by increasing LPS binding to the cells. The binding of 3H-LPS to HUVEC was determined in the absence or presence of Hb or two other known LPS-binding proteins, human serum albumin (HSA) and IgG. LPS binding was substantially increased in the presence of Hb, in a Hb concentration-dependent manner, but was not increased by HSA or IgG. Hb enhancement of LPS binding was observed in serum-free medium, indicating that there was no additional requirement for any of the serum factors known to participate in the interaction of LPS with cells (e.g., lipopolysaccharide (LPS)-binding protein (LBP) and soluble CD14 (sCD14)). Hb enhancement of LPS binding also was observed in the more physiologic condition of 100% plasma. LPS-induced TF activity was stimulated by Hb, but not by HSA or IgG. In serum-free medium, TF activity was not stimulated under any of the conditions tested. Ultrafiltration of LPS was dramatically increased after incubation with Hb but not with HSA or IgG, suggesting that LPS disaggregation by Hb was responsible for the enhanced binding of LPS to HUVEC and the subsequent stimulation of TF activity.

THE INFLUENCE OF SERUM ON THE UPTAKE, CONVERSION AND ACTION OF DIHYDROTESTOSTERONE IN RAT PROSTATE GLANDS IN ORGAN CULTURE

1975 ◽  
Vol 64 (2) ◽  
pp. 289-297 ◽  
Author(s):  
ILSE LASNITZKI ◽  
HILARY R. FRANKLIN
Keyword(s):  
Organ Culture ◽  
Horse Serum ◽  
Target Tissue ◽  
Serum Free Medium ◽  
Free Medium ◽  
Serum Free ◽  
Human Pregnancy ◽  
Human Male ◽  
Columnar Cells

SUMMARY The influence of serum on the uptake, conversion and action of dihydrotestosterone in relation to the sex steroid binding protein, TeBG, has been investigated in rat ventral prostates in organ culture. The organs were incubated with [1,2-3H]dihydrotestosterone in: (1) serum-free medium, (2) horse serum, foetal and newborn bovine serum or (3) human male and human pregnancy serum. With all sera the uptake of dihydrotestosterone fell with rising serum concentration, at first steeply and then more gradually. At the same concentration, the uptake was significantly lower in explants incubated with human pregnancy serum than in those kept with human male serum. The conversion of dihydrotestosterone to androstanediol followed the same pattern and less androstanediol was formed in the presence of pregnancy serum. Since pregnancy serum contains higher amounts of TeBG than male serum, the lowered uptake suggests that only the free hormone was available to the target organ. Addition of unlabelled dihydrotestosterone resulted in a higher uptake than that measured in explants incubated with the labelled steroid only. The effect of the human sera on uptake and conversion was correlated with the androgenic activity of dihydrotestosterone applied at physiological concentrations and expressed as the percentage of secretory columnar cells present. The degree of maintenance closely corresponded to the uptake of the hormone. In serum-free medium, the number of columnar cells approached the values found in vivo, with male serum their number, though reduced, was still substantial, with pregnancy serum it was extremely low. It is concluded that the amounts of TeBG present in serum regulate the supply of the hormone to the target tissue and thus control its biological action.

scholarly journals The effects of various hormones and growth factors on the growth of human insulin-producing cell line in serum-free medium

1997 ◽  
Vol 29 (4) ◽  
pp. 209-216 ◽  
Author(s):  
Jae-Jeong Lee ◽  
Jai-Hyun Kwon ◽  
Yong Keun Park ◽  
Ohoak Kwon ◽  
Tai-Wook Yoon
Keyword(s):  
Growth Factors ◽  
Cell Line ◽  
Human Insulin ◽  
Serum Free Medium ◽  
Free Medium ◽  
Serum Free

Therapeutic Impact of Human Bone Marrow Stromal Cells Expanded by Animal Serum–Free Medium for Cerebral Infarct in Rats

Neurosurgery ◽  
2011 ◽  
Vol 68 (6) ◽  
pp. 1733-1742 ◽  
Author(s):  
Taku Sugiyama ◽  
Satoshi Kuroda ◽  
Yukari Takeda ◽  
Mitsufumi Nishio ◽  
Masaki Ito ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Marrow Stromal Cells ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Serum Free Medium ◽  
Free Medium ◽  
Serum Free ◽  
Therapeutic Impact
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