A Kinetic Stem Cell Counting Analysis of the Specific Effects of Cell Culture Medium Growth Factors on Adipose-Derived Mesenchymal Stem Cells

Abstract

A recently described kinetic stem cell (KSC) counting method was used to investigate the stem-cell-specific effects of commercial growth factor supplements used for expanding stem cells in adipose-tissue-derived mesenchymal cell preparations. The supplements were a proprietary growth factor product, a source of fetal bovine serum, two sources of pooled human sera, and two sources of human platelet lysate. KSC counting analyses were performed to monitor effects on the fraction and viability of stem cells in serial cultures with their respective supplements. Serial cultures supplemented with the proprietary growth factor product or fetal bovine serum showed a similar high degree of maintenance of stem cell fraction with passage. In contrast, cultures supplemented with human sera or human platelet lysate showed rapid declines in stem cell fraction. KSC counting was used to discover the cellular basis for the decreasing stem cell fractions. For human platelet lysate, it was attributable to lower rates of self-renewing symmetric stem cell divisions. For human sera, both low rates of symmetric division and high rates of stem cell death were responsible. These results demonstrate the power of the KSC counting method to provide previously inaccessible information for improving future tissue stem cell biomanufacturing.

Quantification of the Culture Stability of Stem Cell Fractions from Oral-Derived, Human Mesenchymal Stem Cell Preparations: A Significant Step toward the Clinical Translation of Cell Therapies

“…Our analyses establish that the SCF of these tissue cell preparations can vary significantly among donors. The KSC counting method also revealed that, unlike previously described human tissue stem-cell-containing preparations [15,16], including MSC-containing preparations from other sources (e.g., bone marrow and adipose tissue) [13,16], the SCF of some donor aBMSC-containing populations is stable with serial cell culture. This newly discovered property may inform future work to establish more effective biomanufacturing processes for MSCs, as well as other tissue stem cell types, for use in SCTs.…”

“…Briefly, 1 × 10 5 cells/well were used to start cultures. Serial cell culture was performed as described previously for KSC counting analyses of MSC-containing tissue cell preparations [13], with the counting of live cells and dead cells at each culture passage based on trypan blue dyeexclusion. Cell counting was performed manually according to well established methods using a hemocytometer slide.…”

“…Serial cell culture of all eight aBMSC strains was conducted in CCM on the same passage schedule, which was a transfer of 1/20 of the total recovered cells every 72 h, within a three-hour range of variance. As required for KSC counting analyses, serial cell cultures were continued until no cells could be detected [13,15].…”

“…Despite these criteria being used widely in the field, in the context of translational studies toward the development of cell therapies, the heterogeneous nature of different MSC-containing populations confounds the understanding of how they can be used. MSC-containing populations from diverse tissue sources (with alveolar bone being one example) are, in fact, heterogeneous mixtures of stem cells, committed progenitor cells, and committed cells [8,[11][12][13][14]. Currently, fluorescence-activated cell sorting (FACS) is used to purify and quantify specific cell populations based on immunophenotypes detected using flow cytometry.…”

“…For these studies, we employed a recently described method called kinetic stem cell (KSC) counting. KSC counting is a computational simulation method that provides the routine, reproducible, and accurate determination of the SCF of heterogeneous organ and tissue cell populations [13,15,16]. Here, we report for the first time the use of KSC counting to determine SCFs in oral-derived aBMSC-containing preparations.…”