Isolation of adipose tissue derived regenerative cells from human subcutaneous tissue with or without the use of an enzymatic reagent

Abstract

Freshly isolated, uncultured, autologous adipose derived regenerative cells (ADRCs) have emerged as a promising tool for regenerative cell therapy. The Transpose RT system (InGeneron, Inc., Houston, TX, USA) is a system for isolating ADRCs from adipose tissue, commercially available in Europe as a CE-marked medical device and under clinical evaluation in the United States. This system makes use of the proprietary, enzymatic Matrase Reagent for isolating cells. The present study addressed the question whether the use of Matrase Reagent influences cell yield, cell viability, live cell yield, biological characteristics, physiological functions or structural properties of the ADRCs in final cell suspension. Identical samples of subcutaneous adipose tissue from 12 subjects undergoing elective lipoplasty were processed either with or without the use of Matrase Reagent. Then, characteristics of the ADRCs in the respective final cell suspensions were evaluated. Compared to non-enzymatic isolation, enzymatic isolation resulted in approximately twelve times higher mean cell yield (i.e., numbers of viable cells/ml lipoaspirate) and approximately 16 times more colony forming units. Despite these differences, cells isolated from lipoaspirate both with and without the use of Matrase Reagent were independently able to differentiate into cells of all three germ layers. This indicates that biological characteristics, physiological functions or structural properties relevant for the intended use were not altered or induced using Matrase Reagent. A comprehensive literature review demonstrated that isolation of ADRCs from lipoaspirate using the Transpose RT system and the Matrase Reagent results in the highest viable cell yield among published data regarding isolation of ADRCs from lipoaspirate.

Fig 1

Frequency distributions of cell yield (A) and cell viability (B) data reported in the literature for enzymatic (green) and non-enzymatic (red) methods for isolating ADRCs from adipose tissue. Interpretation of this data is illustrated by the following examples: (i) For six out of ten non-enzymatic methods (60%) cell yield between 0 and 1×10⁵ cells per ml lipoaspirate was reported (“a” in Panel A). (ii) For nine out of 39 enzymatic methods (21%) cell yield between 2×10⁵ and 3×10⁵ cells per ml lipoaspirate was reported (“b” in Panel A). (iii) For only one out of ten non-enzymatic methods (10%) cell viability was reported (between 60.1% and 70%) (“c” in Panel B). (iv) For ten out of 39 enzymatic methods (20.5%) cell viability between 80.1% and 90% was reported (“d” in Panel B). (v) Unfortunately, for nine out of ten non-enzymatic methods (90%) as well as for 14 out of 39 enzymatic methods (36%) no cell viability data were reported (“e” in Panel B).

Fig 2. Isolation of ADRCs from human adipose tissue with the Transpose RT system and the Matrase Reagent (both from InGeneron, Inc., Houston, TX, USA).

Details are provided in the text.

Fig 3. Results of quantitative analysis of Transpose RT / no Matrase isolated ADRCs and Transpose RT / Matrase isolated ADRCs.

The panels show Tukey boxplots of the number of cells isolated per ml lipoaspirate (A), the relative number of viable cells (B), the number of viable cells isolated per ml lipoaspirate (C) and the diameter of cells (D) of Transpose RT / no Matrase isolated ADRCs (red bars) and Transpose RT / Matrase isolated ADRCs (green bars). In (B) the threshold of 70% viable cells established by the International Federation for Adipose Therapeutics and Science (IFATS) [73] is indicated by a dashed line. Results of Wilcoxon matched-pairs signed rank test are indicated (n = 12 paired samples each). ***, p < 0.001.

Fig 4. Results of colony forming unit assay.

The panel shows Tukey boxplots of the number of colony forming units per ml lipoaspirate formed by ASCs derived from Transpose RT / no Matrase isolated ADRCs (red bars) and from Transpose RT / Matrase isolated ADRCs (green bars) after culturing for 14 days in complete MSC media. Results of Wilcoxon matched-pairs signed rank test are indicated (n = 10 paired samples). **, p < 0.005.

Fig 5. Formation of embryoid bodies.

The panels show embryoid bodies that were formed after culturing ASCs derived from Transpose RT / no Matrase isolated ADRCs (A, C) and from Transpose RT / Matrase isolated ADRCs (B, D) for seven days in serum-free media. The scale bar in (D) represents 100 μm in (A, B) and 50 μm in (C, D).

Fig 6. Quantitative analysis of the size of embryoid bodies.

The panel shows Tukey boxplots of the diameter of embryoid bodies that were formed after culturing ASCs derived from Transpose RT / no Matrase isolated ADRCs (red bars) and from Transpose RT / Matrase isolated ADRCs (green bars) for seven days in serum-free media. The Wilcoxon matched-pairs signed rank test showed no statistically significant differences between the groups (p = 0.109; n = 4 paired samples).

Fig 7. Results of gene expression analysis.

The panels show Tukey boxplots of relative gene expression (arbitrary units) of Oct4 (A, B), Klf4 (C, D) and Hes3 (E, F) of ASCs in conventional monolayer cultures (A, C, E) or obtained from embryoid bodies (B, D, F) after culturing Transpose RT / no Matrase isolated ADRCs (red bars) or Transpose RT / Matrase isolated ADRCs (green bars), respectively. The Wilcoxon matched-pairs signed rank test showed no statistically significant differences between the groups (p > 0.05; n = 8 paired samples each).

Fig 8. Adipogenic differentiation potential of ADRCs.

The panels show the results of culturing ASCs on their 3^rd passage (derived from Transpose RT / no Matrase isolated ADRCs (A, C) or Transpose RT / Matrase isolated ADRCs (B, D), respectively) for two weeks in adipogenic differentiation medium (A, B) or control medium (C, D). The presence of intracytoplasmic lipids (triglycerides) was assessed with Oil red-O staining; cells were counterstained with hematoxylin. The yellow arrows indicate single Oil red-O positive cells. The scale bar in (D) represents 100 μm in (A-D).

Fig 9. Quantitative analysis of adipogenic differentiation potential of ADRCs.

The panel shows Tukey boxplots of the relative number of Oil red-O positive cells obtained after culturing ASCs on their 3rd passage (derived from Transpose RT / no Matrase isolated ADRCs (red bars) or Transpose RT / Matrase isolated ADRCs (green bars), respectively) for two weeks in adipogenic differentiation medium. The Wilcoxon matched-pairs signed rank test showed no statistically significant differences between the groups (p = 0.109; n = 7 paired samples).

Fig 10. Osteogenic differentiation potential of ADRCs.

The panels show the results of culturing ASCs on their 3^rd passage (derived from Transpose RT / no Matrase isolated ADRCs (A, C) or Transpose RT / Matrase isolated ADRCs (B, D), respectively) for two weeks in osteogenic differentiation medium (A, B) or control medium (C, D). The presence of calcific deposits was investigated with Alizarin red staining; cells were counterstained with hematoxylin. Cells of an osteogenic lineage are stained bright to deep red and easily visible as dense red patches. The scale bar in (D) represents 100 μm in (A-D).

Fig 11. Hepatogenic differentiation potential of ADRCs.

The panels show the results of culturing ASCs on their 3^rd passage (derived from Transpose RT / no Matrase isolated ADRCs (A, C, E) or Transpose RT / Matrase isolated ADRCs (B, D, F), respectively) for ten days in hepatogenic differentiation medium (A-D) or control medium (E, F). The presence of structures containing a high proportion of carbohydrate macromolecules (glycogen, glycoprotein and proteoglycans) was investigated with Periodic Acid Schiff staining; cells were counterstained with hematoxylin. As a result of induction of hepatogenesis the morphology of the cells changed from a fibroblastic spindle shape to a rather polygonal shape typically associated with hepatocytes. The scale bar in (F) represents 100 μm in (A, B, E, F) and 50 μm in (C, D).

Fig 12. Neurogenic differentiation potential of ADRCs (1).

The panels show the results of culturing ASCs on their 6^th passage (derived from Transpose RT / no Matrase isolated ADRCs (A, C) or Transpose RT / Matrase isolated ADRCs (B, D), respectively) for three weeks in neurogenic differentiation medium (A, B) or control medium (C, D). Cells were imaged with phase contrast microscopy. As a result of induction of neurogenesis, the cells developed characteristic, slender processes (arrows in A, B). The scale bar in (D) represents 50 μm in (A-D).

Fig 13. Neurogenic differentiation potential of ADRCs (2).

The panels show the results of culturing ASCs on their 6^th passage (derived from Transpose RT / no Matrase isolated ADRCs (A, C, E, G) or Transpose RT / Matrase isolated ADRCs (B, D, F, H), respectively) for three weeks in neurogenic differentiation medium (A, B, E, F) or control medium (C, D, G, H). Cells were processed with immunofluorescence for the detection of respectively microtubule-associated protein 2 (MAP2) (A-D) or beta III Tubulin (β3TUB) (E-H), and were counterstained with DAPI. As a result of induction of neurogenesis, the cells expressed both MAP2 (A, B) and β3TUB (E, F). The scale bar in (H) represents 50 μm in (A-H).

Fig 14. Expression of microtubule-associated protein 2 (MAP2) and beta III tubulin (β3TUB) by PC12 cells as positive control.

The panels show the results of culturing PC12 cells for 6 days in neurogenic culture medium. Cells were processed with immunofluorescence for the detection of respectively MAP2 (A) or β3TUB (B), and were counterstained with DAPI. The scale bar in (B) represents 50 μm in (A, B).

Fig 15. Measurement of collagenase activity in cell preparations that were prepared with the use of Matrase Reagent following the manufacturer’s instructions for use.

No collagenase activity was detected in cell preparations (the red dot at Y = X = 0 [indicated by the red arrow] represents the mean of two independent measurements for each sample). The standard curve for the used assay (EnzChek Gelatinase/Collagenase Assay Kit E12055; Invitrogen) is depicted by the green line. Because this high-sensitivity assay can detect enzyme activity down to a minimum concentration of 2×10⁻³ U/ml, activity of the enzyme in the final cell preparation was not present or less than two-thousands of a unit per ml.

Fig 16. Cell yield and live cell yield obtained with Transpose RT / Matrase isolation of ADRCs and Transpose RT / no Matrase isolation of ADRCs compared with corresponding data reported in the literature.

Panels A and B show individual data as well as mean ± standard error of the mean of cell yield (A) and live cell yield (B) obtained with Transpose RT / Matrase isolation of ADRCs (black dots), enzymatic methods for isolating ADRCs reported in the literature (listed in Table 1) (green dots), Transpose RT / no Matrase isolation of ADRCs (black crosses) and non-enzymatic methods for isolating ADRCs reported in the literature (listed in Table 2) (red dots). Panels C and D show the same data as a function of the year of publication.