Review for "A functional long‐term 2D serum‐free human hepatic <i>in vitro</i> system for drug evaluation"

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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Utilising FGF2, IGF2 and FSH in serum-free protocol for long-term in vitro cultivation of primary human granulosa cells

Molecular and Cellular Endocrinology ◽

10.1016/j.mce.2020.110816 ◽

2020 ◽

Vol 510 ◽

pp. 110816

Author(s):

Kati Hensen ◽

Martin Pook ◽

Anu Sikut ◽

Tõnis Org ◽

Toivo Maimets ◽

...

Keyword(s):

Granulosa Cells ◽

In Vitro Cultivation ◽

Serum Free ◽

Human Granulosa Cells

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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 ◽

10.1182/blood.v83.1.128.128 ◽

1994 ◽

Vol 83 (1) ◽

pp. 128-136 ◽

Cited By ~ 106

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.

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Quantitation of the quiescent fraction of long-term culture-initiating cells in normal human blood and marrow and the kinetics of their growth factor-stimulated entry into S-phase in vitro

Blood ◽

10.1182/blood.v86.9.3314.bloodjournal8693314 ◽

1995 ◽

Vol 86 (9) ◽

pp. 3314-3321 ◽

Cited By ~ 70

Author(s):

L Ponchio ◽

E Conneally ◽

C Eaves

Keyword(s):

Specific Activity ◽

Hematopoietic Cells ◽

High Specific Activity ◽

S Phase ◽

Serum Free Medium ◽

Free Medium ◽

Serum Free ◽

Long Term Culture

A method for quantitating the proportion of cycling long-term culture- initiating cells (LTC-IC) in heterogeneous populations of human hematopoietic cells is described. This procedure involves incubating the cells of interest for 16 to 24 hours in a serum-free medium containing 100 ng/mL Steel factor (SF), 20 ng/mL interleukin-3 (IL-3), and 20 ng/mL granulocyte-colony-stimulating factor (G-CSF), with or without 20 microCi/mL of high specific activity 3H-thymidine (3H-Tdr) before plating the recovered cells in standard LTC-IC assays. The details of this procedure are based in part on the finding that the number of LTC-IC (regardless of their cycling status) remains constant for at least 24 hours under these culture conditions, as long as 3H-Tdr is not present. In addition, we have determined that a 16-hour period of exposure to the 3H-Tdr is sufficient to maximize the discrimination of cycling LTC-IC but not long enough to allow a detectable redistribution of LTC-IC between noncycling and cycling compartments. Finally, any isotope reutilization that may occur is not sufficient to affect the LTC-IC 3H-Tdr suicide values measured. Application of this methodology to normally circulating LTC-IC showed these to be a primarily quiescent population. However, within 72 hours of incubation in a serum-free medium containing SF, IL-3, and G-CSF, most had entered S-phase, although there was no net change in their numbers. This suggests that, under certain conditions in vitro, self-renewal divisions of LTC-IC can occur and, at least initially, balance any losses of these cells due to their differentiation or death. In contrast, many of the LTC-IC in freshly aspirated samples of normal marrow were found to be proliferating, although those that were initially quiescent could also be recruited into S-phase within 72 hours in vitro when incubated under the same conditions used to stimulate circulating LTC-IC. This modified 3H-Tdr suicide procedure should facilitate further investigation of the mechanisms regulating the turnover of the most primitive compartments of human hematopoietic cells and how these may be altered in disease states or exploited for a variety of therapeutic applications.

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