Adipose-Derived Stromal/Stem Cell Culture: Effects of Different Concentrations of Human Platelet Lysate in Media

2020 ◽  
pp. 1-9
Author(s):  
Olga R. Ballesteros ◽  
Patrick T. Brooks ◽  
Eva K. Haastrup ◽  
Anne Fischer-Nielsen ◽  
Lea Munthe-Fog ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cell ◽  
Chemokine Receptors ◽  
Human Platelet ◽  
Platelet Lysate ◽  
Population Doubling ◽  
T Cell Proliferation ◽  
Growth Media ◽  
Wide Range ◽  
Human Platelet Lysate

Adipose-derived stromal/stem cells (ASCs) are being tested as a possible treatment for a wide range of diseases to exploit the immunomodulatory and regenerative potential demonstrated in vitro. Pooled human platelet lysate (pHPL) has replaced fetal bovine serum (FBS) as the preferred growth supplement because of its xeno-free origin and improved cell proliferation. Much has been done toward reducing the concentration of pHPL required when expanding ASCs. However, little is known on how increasing the concentration of pHPL affects ASC potency, which could lead to changes with possible beneficial applications. This study investigated the effect of 5, 10, or 20% pHPL in culture media on ASC proliferation and phenotypic marker expression, including chemokine receptors CXCR2, CXCR3, CXCR4, and VLA-4. Adipogenic and osteogenic properties, as well as immunosuppressive properties, including the ability to induce indoleamine-pyrrole 2,3-dioxygenase 1 (IDO1) and suppress T cell proliferation, were also examined. We observed a significant increase in cell yield (approximately 2-fold) and a corresponding reduction in population doubling time and cell volume when doubling the concentration of pHPL in the growth media. ASCs maintained expression of phenotypic surface markers CD73, CD90, and CD105 and were negative for CD45 and CD31. The ability to induce IDO1 and suppress T cell proliferation was observed as well. Adipogenesis and osteogenesis, however, seem to be increased at higher concentrations of pHPL (20% > 10% > 5%), while expression of chemokine receptors CXCR2 and CXCR3 was lower. In conclusion, increasing the pHPL concentration to 20% could be used to optimize culture conditions when producing cells for clinical treatments and may even be used to enhance beneficial ASC properties depending on the desired therapeutic effect.

scholarly journals Human Platelet Lysate Media Supplement Supports Lentiviral Transduction and Expansion of Human T Lymphocytes While Maintaining Memory Phenotype

2019 ◽  
Vol 2019 ◽  
pp. 1-11 ◽  
Author(s):  
Emanuele Canestrari ◽  
Hayley R. Steidinger ◽  
Brianna McSwain ◽  
Steven J. Charlebois ◽  
Christina Tenenhaus Dann
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Lymphocytes ◽  
Human Platelet ◽  
Interleukin 2 ◽  
Platelet Lysate ◽  
Central Memory ◽  
Protein Profiling ◽  
Memory Phenotype ◽  
Human Platelet Lysate

Immune cell therapy has emerged as a promising approach to treat malignancies that were up until recently only treated on a palliative basis. Chimeric antigen receptor- (CAR-) modified T lymphocytes (T cells) in particular have proven to be very effective for certain hematological malignancies. The production of CAR T cells usually involves viral transduction andex vivoculture of T cells. The aim of this study was to explore the use of human platelet lysate (HPL) compared to two commonly used supplements, human AB serum (ABS) and fetal bovine serum (FBS), for modified T cell production. For studying transduction, activated T cells were transduced with lentivirus to deliver GFP transgenes with three different promoters. Transduction efficiency (percent GFP) was similar among the supplements, and a modest increase in the transgene product (mean fluorescence intensity) was observed when HPL was used as a supplement compared to ABS. To study the effect of supplements on expansion, peripheral blood mononuclear cells (PBMCs) were activated and expanded in the presence of interleukin 2 (IL2) for fourteen days. T cell expansions using HPL and ABS were comparable and slightly less than the expansion obtained with FBS. Interestingly, cells expanded in media supplemented with HPL showed a higher percentage of T cells with a central memory phenotype compared to those expanded in ABS or FBS. Protein profiling revealed that the phenotypic differences may be explained by elevated levels of several cytokines in HPL, including IL7. The results suggest that the use of HPL as a cell culture supplement during the production of modified T cells is a reasonable alternative to ABS. Furthermore, the use of HPL may enhancein vivoperformance of the final product by enriching for central memory T cells that are associated with long-term persistence following adoptive transfer.

scholarly journals Immunogenicity and Protective Activity of Pigeon Circovirus Recombinant Capsid Protein Virus-Like Particles (PiCV rCap-VLPs) in Pigeons (Columba livia) Experimentally Infected with PiCV

Vaccines ◽  
2021 ◽  
Vol 9 (2) ◽  
pp. 98
Author(s):  
Huai-Ying Huang ◽  
Benji Brayan I. Silva ◽  
Shen-Pang Tsai ◽  
Ching-Yi Tsai ◽  
Yu-Chang Tyan ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cell ◽  
Capsid Protein ◽  
Expression System ◽  
Genetic Material ◽  
Columba Livia ◽  
T Cell Proliferation ◽  
Virus Like Particles ◽  
Mammalian Expression ◽  
Wide Range

Pigeon circovirus (PiCV) is the most recurrent virus diagnosed in pigeons and is among the major causative agents of young pigeon disease syndrome (YPDS). Due to the lack of an established laboratory protocol for PiCV cultivation, development of prophylaxis is hampered. Alternatively, virus-like particles (VLPs), which closely resemble native viruses but lack the viral genetic material, can be generated using a wide range of expression systems and are shown to have strong immunogenicity. Therefore, the use of VLPs provides a promising prospect for vaccine development. In this study, transfected human embryonic kidney (HEK-293) cells, a mammalian expression system, were used to express the PiCV capsid protein (Cap), which is a major component of PiCV and believed to contain antibody epitopes, to obtain self-assembled VLPs. The VLPs were observed to have a spherical morphology with diameters ranging from 12 to 26 nm. Subcutaneous immunization of pigeons with 100 µg PiCV rCap-VLPs supplemented with water-in-oil-in-water (W/O/W) adjuvant induced specific antibodies against PiCV. Observations of the cytokine expression and T-cell proliferation levels in spleen samples showed significantly higher T-cell proliferation and IFN- γ expression in pigeons immunized with VLPs compared to the controls (p < 0.05). Experimentally infected pigeons that were vaccinated with VLPs also showed no detectable viral titer. The results of the current study demonstrated the potential use of PiCV rCap-VLPs as an effective vaccine candidate against PiCV.

scholarly journals Potential Effect of Human Platelet Lysate on in vitro Expansion of Human Corneal Endothelial Cells Compared with Y-27632 ROCK Inhibitor

2021 ◽  
Author(s):  
Mohammad Amir Mishan ◽  
Sahar Balagholi ◽  
Tahereh Chamani ◽  
Sepehr Feizi ◽  
Zahra-Soheila Soheili ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Endothelial Cells ◽  
Human Platelet ◽  
Platelet Lysate ◽  
Corneal Endothelial Cells ◽  
Rock Inhibitor ◽  
Treatment Groups ◽  
Medium Supplement ◽  
Proliferative Effect ◽  
Human Platelet Lysate

Purpose: Corneal endothelial cell (CEC) therapy can be used as a promising therapeutic option for patients with various corneal endothelial dysfunctions. In this study, we compared the proliferative effect of human platelet lysate (HPL), as a xeno-free medium supplement, with Y-27632 Rho/rho-associated protein kinase (ROCK) inhibitor, as a wellknown proliferative and adhesive agent for CECs, and fetal bovine serum (FBS) as the control, in the culture medium of human corneal endothelial cells (HCECs). Methods: We isolated HCECs from human donors and treated the cells as three different treatment groups including 20% HPL only, 10 μM Y-27632 ROCK inhibitor, combination of 20% HPL and 10 μM Y-27632 ROCK inhibitor, and 20% FBS as the control group. ELISA cell proliferation assay and cell counting was performed on the treated cells. Finally, HCECs were characterized by morphology and immunocytochemistry (ICC). Results: There was no significant proliferative effect of HPL on cell proliferation compared with the cells treated with Y-27632 ROCK inhibitor or the combination of HPL and Y-27632 ROCK inhibitor, but all the respected treatments had significant inducible effect on cell proliferation as compared with FBS-treated cells. The cells grown in all three treatment groups exhibited CEC morphology. Also, there was a higher expression of Na+/K+-ATPase and ZO-1, as CEC characteristic markers, in the culture of HCECs treated with HPL as compared with FBS. Conclusion: HPL offers a xeno−free and affordable medium supplement for CEC expansion that can be used in clinical applications.

Identification of Critical Amino Acids in Human Platelet Factor 4 (PF4) Required for Its Modulatory Effects on CD4+CD25+ Regulatory T Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1339-1339
Author(s):  
Chao Yan Liu ◽  
Rhonda A. Geoffrey ◽  
Qi-Hong Sun ◽  
Gian Paolo Visentin
Keyword(s):  
Amino Acids ◽  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Human Platelet ◽  
T Cell Proliferation ◽  
Heparin Binding ◽  
Wild Type ◽  
Platelet Factor ◽  
N Terminus

Abstract Human platelet factor 4 (PF4; CXCL4), a heparin-binding CXC chemokine contained in platelet α-granules, is secreted upon activation of platelets. Several reports have identified PF4 as an inhibitor of hematopoietic progenitor and endothelial cell proliferation and angiogenesis. Furthermore, PF4 has been shown to strongly inhibit T cell proliferation as well as IFN-γ and IL-2 release by activated T cells. We have recently reported that human PF4 inhibits the proliferative response of human CD4+CD25− T cells, while inducing expansion of CD4+CD25+ T regulatory (Tr) cells stimulated by anti-CD3 or anti-CD3 and anti CD-28 monoclonal antibodies (mAbs), and that PF4-induced CD4+CD25+ Tr cells lose their potent suppressor function in vitro. The antithetic effects of PF4 on CD4+CD25− T cells and CD4+CD25+ Tr cells in response to anti-CD3 and anti-CD3/28 mAbs appear to be specific since these effects were not mimicked by equivalent quantities of protamine, a positively charged heparin-binding protein used as a control for any putative interaction mediated only by positive charges, or heparin alone. We hypothesize that PF4 acts not only as the target for the antibody, in patients experiencing heparin-induced thrombocytopenia (HIT), but also as a modulator of T regulatory lymphocytes. How PF4 modulates T cell proliferation and CD4+CD25+ Tr cell-mediated suppression of CD4+CD25− T cells remains to be determined. To delineate which domain(s) of PF4 is(are) critical for its antithetical activity on the two T cell subsets, we compared PF4 from rat and cattle, which are about 74% homologous to human PF4, for their ability to affect proliferation of CD4+CD25+ and CD4+CD25− T cells stimulated by anti-CD3 mAb. Both rat and bovine PF4 were recognized by a rabbit polyclonal Ab against human PF4, but neither one, in contrast with human PF4, was capable to induce proliferation of CD4+CD25+ Tr cells. This suggests that the relatively few amino acid (AA) residues at which rat and human differ are critical for the modulatory activity of PF4 on T cells. Therefore, we introduced specific modifications into human PF4 cDNA using site-directed mutagenesis to determine the importance of individual AA residues for PF4 effects on T cell proliferation. Our strategy involved converting selected amino acids in human PF4 (reactive) to the corresponding residues in rat PF4 (non-reactive) and determining the effect of each change on the proliferation of CD4+CD25+ and CD4+CD25− T cells stimulated by anti-CD3 mAb. We created seven mutant forms of PF4. Four of the mutants involved one or more of six residues in the 47 C-terminal AAs, known to comprise the most important heparin binding region. The other three mutants were created at the N-terminus. All the PF4 constructs, expressed in E. coli, assembled properly into tetramers as determined by their HPLC profile and electrophoretic mobility in SDS-PAGE and were comparable to wild-type PF4 in their avidity for heparin. PF4 constructs bearing mutations at the N-terminus (E4S, L11V, and T16S) and three C-terminal mutants (the combined P37A/T38V/A39P, R49S, and L55R) were comparable to wild type recombinant human PF4 for their proliferative effects on the two T cell subpopulations. In contrast, another C-terminal mutant (A57V) completely abrogated PF4 proliferative effects on CD4+CD25+ Tr cells, suggesting the importance of this determinant in the interaction with a specific receptor on T cells.

scholarly journals Human Platelet Lysate (HPL) as an Alternative Media Propagation of T47D Cells Line

2020 ◽  
Vol 11 (1) ◽  
pp. 36
Author(s):  
Diani Mentari ◽  
Relita Pebrina ◽  
Diah Nurpratami
Keyword(s):  
Cell Proliferation ◽  
Total Protein ◽  
Doubling Time ◽  
Culture Media ◽  
Human Platelet ◽  
T47d Cell ◽  
Platelet Lysate ◽  
Blood Type ◽  
Human Platelet Lysate ◽  
Cell Propagation

Fetal bovine serum (FBS) is a gold standard as a supplement to cell and tissue culture media. This is due to a large number of Growth Factor (GF) contained in FBS. However, the use of FBS is at risk of transferring endotoxins, prions, bacteria and viruses from animals to humans, so it is risky to be used on cell therapy. Human Platelet Lysate (HPL) is a medium that can be developed as an alternative cell growth medium. The advantage of HPL is that it does not contain aggregate platelets so it does not cause the cells to clot. This condition causes HPL to be used as a substitute medium replacing FBS for cell propagation. The use of HPL for cell propagation has been widely reported. However, the use of HPL in cancer cells has not been found. Thus, this study aims to see the effectiveness of HPL as a T47D cell culture medium. The study began with donor selection with criteria for the male sex, the blood type O, the age ≤35 years. Furthermore, the Platelet Concentrate (PC) was processed into HPL then measured pH, total protein and albumin levels. The cell viability was measured using the MTT assay to determine the ability of cell proliferation when propagation using HPL. The doubling time test was carried out as in the cell proliferation test. However, the incubation was carried out for 24 h, 48 h and 72 h and the HPL concentration used was 5%. The result shows that HPL 10% and 20% ability to increase proliferation better than the FBS 10%. HPL with a 5% concentration ability to shortens the doubling time than FBS 10% (doubling time is less than 19.94 h). It this study, cell proliferation is influenced by the pH of HPL and total protein but not by the amount albumin.Keywords: Human Platelet Lysate, Proliferation, T47D cell line, total protein, albumin.

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