A functional long‐term 2D serum‐free human hepatic in vitro system for drug evaluation

2020 ◽  
Author(s):  
Carlota Oleaga ◽  
L. Richard Bridges ◽  
Keisha Persaud ◽  
Christopher W. McAleer ◽  
Christopher J. Long ◽  
...  
Keyword(s):  
Drug Evaluation ◽  
In Vitro System ◽  
Serum Free

scholarly journals Amplification of Sca-1+ Lin-WGA+ cells in serum-free cultures containing steel factor, interleukin-6, and erythropoietin with maintenance of cells with long-term in vivo reconstituting potential

Blood ◽  
1994 ◽  
Vol 83 (1) ◽  
pp. 128-136 ◽  
Author(s):  
VI Rebel ◽  
W Dragowska ◽  
CJ Eaves ◽  
RK Humphries ◽  
PM Lansdorp
Keyword(s):  
Interleukin 6 ◽  
Cell Number ◽  
In Vitro System ◽  
Serum Free ◽  
Murine Bone Marrow ◽  
Steel Factor ◽  
Parallel Increase

Abstract Normal murine bone marrow (BM) cells were sorted on the basis of low forward and orthogonal light scatter properties, Sca-1 expression (Sca-1+), lack of staining with a cocktail of mature hematopoietic lineage markers (Lin-), and binding of wheat germ agglutinin (WGA+). This approach allowed the reproducible isolation of a very small subpopulation (0.037% +/-0.023% of all nucleated BM cells) that was approximately 400-fold enriched in cells capable of reconstituting both lymphoid and myeloid lineages in lethally irradiated recipients. Transplantation of 30 or 10 of these Sca-1+Lin-WGA+ cells resulted in > or = to 20% donor-derived nucleated peripheral blood cells 3 months posttransplantation in 100% and 22% of the recipients, respectively. When Sca-1+Lin-WGA+ cells were cultured in serum-free medium supplemented with Steel factor, interleukin-6 (IL-6), and erythropoietin (with or without IL-3), a large increase in total cell number, including cells with an Sca-1+Lin-WGA+ phenotype was observed. Single cell cultures showed that 90% to 95% of the input cells underwent at least one division during the first 2 weeks and the remainder died. Interestingly, this proliferative response was not accompanied by a parallel increase in the number of cells with both lymphoid and myeloid repopulating potential in vivo, as quantitation of these by limiting dilution analysis showed they had decreased slightly (1.3-fold) but not significantly below the number initially present. These results demonstrate that Sca-1+Lin-WGA+ cells with long-term repopulating potential can be maintained for 2 weeks in a serum-and stroma cell-free culture, providing a simple in vitro system to study their behavior under well-defined conditions. The observed expansion of Sca-1+Lin-WGA+ cells in vitro without a concomitant increase in reconstituting cells also shows that extensive functional heterogeneity exists within populations of cells with this surface phenotype.

Author response for "A functional long‐term 2D serum‐free human hepatic in vitro system for drug evaluation"

2020 ◽  
Author(s):  
Carlota Oleaga ◽  
L. Richard Bridges ◽  
Keisha Persaud ◽  
Christopher McAleer ◽  
Christopher Long ◽  
...  
Keyword(s):  
Drug Evaluation ◽  
Author Response ◽  
Serum Free

scholarly journals Amplification of Sca-1+ Lin-WGA+ cells in serum-free cultures containing steel factor, interleukin-6, and erythropoietin with maintenance of cells with long-term in vivo reconstituting potential

Blood ◽  
1994 ◽  
Vol 83 (1) ◽  
pp. 128-136 ◽  
Author(s):  
VI Rebel ◽  
W Dragowska ◽  
CJ Eaves ◽  
RK Humphries ◽  
PM Lansdorp
Keyword(s):  
Interleukin 6 ◽  
Cell Number ◽  
In Vitro System ◽  
Serum Free ◽  
Murine Bone Marrow ◽  
Steel Factor ◽  
Parallel Increase

Normal murine bone marrow (BM) cells were sorted on the basis of low forward and orthogonal light scatter properties, Sca-1 expression (Sca-1+), lack of staining with a cocktail of mature hematopoietic lineage markers (Lin-), and binding of wheat germ agglutinin (WGA+). This approach allowed the reproducible isolation of a very small subpopulation (0.037% +/-0.023% of all nucleated BM cells) that was approximately 400-fold enriched in cells capable of reconstituting both lymphoid and myeloid lineages in lethally irradiated recipients. Transplantation of 30 or 10 of these Sca-1+Lin-WGA+ cells resulted in > or = to 20% donor-derived nucleated peripheral blood cells 3 months posttransplantation in 100% and 22% of the recipients, respectively. When Sca-1+Lin-WGA+ cells were cultured in serum-free medium supplemented with Steel factor, interleukin-6 (IL-6), and erythropoietin (with or without IL-3), a large increase in total cell number, including cells with an Sca-1+Lin-WGA+ phenotype was observed. Single cell cultures showed that 90% to 95% of the input cells underwent at least one division during the first 2 weeks and the remainder died. Interestingly, this proliferative response was not accompanied by a parallel increase in the number of cells with both lymphoid and myeloid repopulating potential in vivo, as quantitation of these by limiting dilution analysis showed they had decreased slightly (1.3-fold) but not significantly below the number initially present. These results demonstrate that Sca-1+Lin-WGA+ cells with long-term repopulating potential can be maintained for 2 weeks in a serum-and stroma cell-free culture, providing a simple in vitro system to study their behavior under well-defined conditions. The observed expansion of Sca-1+Lin-WGA+ cells in vitro without a concomitant increase in reconstituting cells also shows that extensive functional heterogeneity exists within populations of cells with this surface phenotype.

Long-Term Dynamic in vitro System for Investigating Rat Pineal Melatonin Secretion

1991 ◽  
Vol 3 (5) ◽  
pp. 563-568 ◽  
Author(s):  
Z. Rékési ◽  
V. Csernus ◽  
J. Horvéth ◽  
S. Vigh ◽  
B. Mess
Keyword(s):  
Melatonin Secretion ◽  
In Vitro System ◽  
Pineal Melatonin

scholarly journals The effect of prior long-term recellularization with keratocytes of decellularized porcine corneas implanted in a rabbit anterior lamellar keratoplasty model

2020 ◽  
Author(s):  
Julia Fernández-Pérez ◽  
Peter W. Madden ◽  
Robert Thomas Brady ◽  
Peter F. Nowlan ◽  
Mark Ahearne
Keyword(s):  
Stromal Cells ◽  
Scar Tissue ◽  
Dendritic Morphology ◽  
Lamellar Keratoplasty ◽  
Human Cornea ◽  
Serum Free ◽  
Anterior Lamellar Keratoplasty ◽  
Potential Alternative

AbstractDecellularized porcine corneal scaffolds are a potential alternative to human cornea for keratoplasty. Although clinical trials have reported promising results, there can be corneal haze or scar tissue. Here, we examined if recellularizing the scaffolds with human keratocytes would result in a better outcome. Scaffolds were prepared that retained little DNA (14.89 ± 5.56 ng/mg) and demonstrated a lack of cytotoxicity by in vitro. The scaffolds were recellularized using human corneal stromal cells and cultured for between 14 in serum-supplemented media followed by a further 14 days in either serum free or serum-supplemented media. All groups showed full-depth cell penetration after 14 days. When serum was present, staining for ALDH3A1 remained weak but after serum-free culture, staining was brighter and the keratocytes adopted a native dendritic morphology with an increase (p < 0.05) of keratocan, decorin, lumican and CD34 gene expression. A rabbit anterior lamellar keratoplasty model was used to compare implanting a 250 µm thick decellularized lenticule against one that had been recellularized with human stromal cells. In both groups, host rabbit epithelium covered the implants, but transparency was not restored after 3 months. Post-mortem histology showed under the epithelium, a less-compact collagen layer, which appeared to be a regenerating zone with some α-SMA staining, indicating fibrotic cells. In the posterior scaffold, ALDH1A1 staining was present in all the acellular scaffold, but in only one of the recellularized lenticules. We conclude that recellularization with keratocytes alone may not be sufficiently beneficial to justify introducing allogeneic cells without concurrent treatment to further manage keratocyte phenotype.

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