Use of animal protein-free products for passaging adherent human adipose-derived stromal/stem cells
Introduction
The potential utility of adipose-derived stromal/stem cells (ASC) has been demonstrated in multiple pre-clinical animal models (1). The ultimate goal is to translate these findings to the bedside through safety and efficacy testing in human subjects. Before conducting such clinical trials, it is necessary to produce human ASC under current good manufacturing practices (cGMP) in accordance with guidelines from the USA Food and Drug Administration (FDA) and the European Medicines Agency (EMA). The use of animal protein-free products will accelerate and facilitate the development of a human ASC product that will meet international approval for clinical use.
Many current human ASC culture protocols employ trypsin isolated from porcine gastric tissue. Indeed, porcine trypsin has been used to dissociate and passage adherent cells, including adipocytes, for decades (2., 3., 4., 5., 6., 7.). Animal protein-derived products potentially present infectious and immunologic concerns in regenerative medicine. Therefore, standard operating procedures (SOPs) that exclude animal protein products need to be developed and validated. There are commercially available substitutes for porcine trypsin. These are a bacterial-derived recombinant product (TrypLE) and a corn-derived product (TrypZean); both are animal protein-free alternatives for cell dissociation and passage. However, there are only a limited number of publications on the use of these products for the passage of embryonic stem cells (ESC), limbic cells and vero cells (8., 9., 10., 11., 12., 13., 14., 15., 16., 17., 18., 19., 20.). While several published studies have employed recombinant bacterial-derived trypsin for the passaging of adherent ASC, no direct comparison of this product relative to porcine trypsin has been presented (9,10,12,13).
This study set out to compare the passage of adherent human ASC using the three sources of trypsin enzyme based on cell yield and viability, differentiation potential of harvested cells, and surface immunophenotype. The findings suggest that no significant differences exist between cells passaged using these trypsin alternatives. Therefore, the present work supports the development of a validated SOP for the passage of human ASC using animal-protein free trypsin product(s).
Section snippets
Donor demographics
Adipose tissue specimens were obtained with informed consent from women undergoing elective liposuction (n = 5). The age range was 24–52 years (mean 44.2 ± 7.9) and body mass index (BMI; weight in kg/height in m2) 21.2–31.1 (mean 25.1 ± 3.9).
ASC isolation, culture, passage and differentiation
ASC were isolated from human subcutaneous adipose lipoaspirate (21). Adherent cells were maintained in stromal medium until 80–90% confluence and harvested by digestion with ready-to-use porcine trypsin (0.05%)/ethyelene diamine tetra acetic acid (EDTA; 1 mm)
Yield and viability
Initial studies compared the yield and viability (Figure 1, top panel) of human ASC passaged with the bacterial-, corn- or porcine-derived trypsin products. The number of recovered cells and their viability were not significantly different among the recombinant-, plant- and porcine-derived trypsin products, at each time-point of enzymatic digestion. Furthermore, the human ASC remained viable for up to 20 min of digestion with all products; cell viability was higher than 95% at time-points of 3–20
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