2.1 Cell Isolation

Human ASCs were harvested from fat tissue obtained by liposuction and abdominoplasties performed at the Department of the authors. Written consent was given by all patients prior to the operation as part of the study's approval by the institutional ethics committee (registration number: 21-0138). Fat tissue sourced from abdominoplasties was minced and placed in a 50 mL tube. We used a modified protocol described by Bunnell et al. [25]. A 0.15% collagenase II-solution (Worthington Biochemical Corp., Lakewood, NJ, USA) in phosphate buffer saline (PBS) (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) was added at a 2:1 ratio to enzymatically digest the tissue. After a 35 min incubation period at 37°C and periodic shaking, the enzymatic reaction was stopped by adding cell culture medium containing foetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). Centrifugation at 1200 g for 10 min led to the formation of three phases including the stromal-vascular-fraction on the bottom, which was used to further isolate the stem cells. The latter was filtered through a 100 μm nylon cell strainer (Falcon), transferred to another tube and subjected to another round of centrifugation at 300 g for 5 min. The cell pellet forming at the bottom of the tube contains the ASC and was resuspended in fresh cell culture medium in a T175 flask (Thermo Fisher Scientific, Waltham, MA, USA) for culture expansion. Cell culture was maintained using Dulbecco's Modified Eagle Medium (DMEM) (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) consisting of 1% (100 μg/mL) Penicillin/Streptomycin (Sigma-Aldrich, Merck Technology, St. Louis, MO, USA), 1% (100 μg/mL) Amphotericin B (Sigma-Aldrich, Merck Technology, St. Louis, MO, USA) and 10% FBS (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) at 37°C and 5% CO₂ in a humidified atmosphere until reaching 80% confluency. Cells were then either cryopreserved in freezing medium or seeded for further experiments.

2.2 Cell Culture

ASC were seeded in biological triplicates on a 12 well-plate at a density of 3 × 10⁴ cells per well in standard cell culture medium +10% FBS. After adhesion, the medium was discarded, the cells were washed with PBS once and the medium was then replaced with the treatment medium. The following four groups were established: (1) standard cell culture medium +10% Platelet-Rich-Fibrin extract (PRFe) (2) standard cell culture medium +10% Platelet-Low-Plasma (3) standard cell culture medium +10% FBS and (4) standard cell culture medium without the addition of any growth supplement (basal medium).

2.3 Flow-Cytometry

According to a joint statement of the international Federation for Adipose Therapeutics and Science (IFATS) and the international Society for Cellular Therapy (ISCT) the following surface markers were used to immunophenotypically characterise the isolated cells at passage 0: CD44, CD29, CD13, CD73, CD90, CD105, CD31, CD45, CD235 (all BioLegend) [26].

Each cell line was seeded in a T75 Flask (Thermo Fisher Scientific, Waltham, MA, USA) until reaching 90% confluency (n = 3). To obtain a single cell solution, cells were trypsinized and maintained in flow-cytometry buffer consisting of 1 mM Ethylenediaminetetraacetic acid (EDTA) (PanReacAppliChem, Darmstadt, Deutschland), 1% bovine serum albumin (BSA) (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and PBS. 1 μL per antibody was then applied to each sample, incubating for 30 min at +4°C.

Control samples included unstained controls and appropriate isotype stain controls.

Finally, samples were run on a BD LSRFortessa Flow Cytometer and the data were analyzed using FlowJo V10.10.0 (BD BioSciences). (Table 1).

2.4 Platelet-Rich-Fibrin-Extract and Platelet-Low-Plasma

Healthy volunteers provided written consent before two vials (10 mL each) of peripheral vein blood were drawn (n = 5). The whole blood samples were centrifuged for 14 min at 1500 rpm without or with (for Platelet-Low-Plasma) the addition of anticoagulants (Sarstedt AG & Co. KG Nuermbrecht, Germany) and rested for an additional 5 min at the bench top [27]. The fibrin polymerised and formed a gel-like substance, residing at the top of the blood collection tube. To separate the erythrocytes at the bottom, the PRF was lifted using forceps (Fuhrmann GmbH, Much, Germany) and cut off 2 mm above the pellet interface. The PRF was then transferred to a 15 mL collection tube and remained at room temperature for 30 min, during which the fibrin clot started to release an initial extract (PRFe). Each vial of blood yielded approximately 1 mL of PRFe and was stored at 4°C until further usage (Figure 1). For cell culture application, the PRFe from these five individual donors was combined and utilised as a pooled supplement.

In contrast to the preparation of PRF, 2 vials (10 mL each) whole blood samples were drawn using blood collection tubes that contained 0.106 mol/L (3.2%) citrate (Sarstedt AG & Co. KG Nuermbrecht, Germany) and centrifuged at 3000 rpm for 10 min [28]. Platelet-Low-Plasma (PLP) was then transferred to a different tube and subjected to an additional round of centrifugation. The PLP was then collected and stored at 4°C until further use. Similar to the PRFe, we used a pooled supplement as our cell culture growth supplement.

2.5 Cell-Viability-Assay

According to the manufacturer's in, struction, the cell Culture medium in each group (n = 3) was removed and replaced with a 10% AlamarBlue Cell Viability Reagent solution (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) diluted in phenol-red-free DMEM (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) at days 1, 3 and 7 of culture. After 2 h of incubation, 100 μL of the supernatant from each well was transferred to a black 96-well plate (Thermo Fisher Scientific, Waltham, MA, USA) and absorption intensity was measured in triplicates at 560 nm (extinction) and 590 nm (emission) in a plate reader (Infinite M Plex, TECAN, Männerdorf, Switzerland). A blank control group was subtracted from each measurement to correct for background signal.

2.6 DNA-Quantification-Assay

Using the protocol provided by the manufacturer, the cell culture medium was discarded and the cells were rinsed with cold PBS. Plated cells were lysed using RLT Buffer (Qiagen, Hilden, Germany) for 10 min on an IKA shaker, and the resulting lysates were collected in 1.5 mL LoBind Tubes (Eppendorf, Hamburg, Germany). 20 μL Proteinase K (PanReacAppliChem, Darmstadt, Deutschland) was added to each tube, followed by another incubation of 10 min at 56°C. We used the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) to extract and purify DNA from the samples, which was pipetted into a black 96 well plate (Thermo Fisher Scientific, Waltham, MA, USA) in 100 μL duplicates (n = 3). The PicoGreen reagent from Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) was diluted in Tris-EDTA buffer to obtain the staining solution, of which 100 μL was added per well. Finally, the fluorescence signal emitted at 550 nm was measured using a plate reader (Infinite M Plex, TECAN, Männerdorf, Switzerland). To quantify the concentration of DNA in our samples, we compared the signal intensity to samples in a standard curve with known concentrations.

2.7 Live-Dead-Assay

At a concentration of 25 mg/mL, FDA (Sigma-Aldrich, Merck Technology, St. Louis, MO, USA) was dissolved in 99.7% Acetone (Carl-Roth GmbH, Karlsruhe, Germany).

Following 7 days of treatment, cell culture medium was discarded, the wells washed with PBS and subsequently, 0,4 μL FDA staining solution was added to 1 mL PBS to incubate in the dark (n = 3). After 5 min, 20 μL of 1 mg/mL PI (Sigma-Aldrich, Merck Technology, St. Louis, MO, USA) staining was added to the incubating staining solution and discarded after 5 s. After two further steps of washing, the cells were imaged using an inverted epifluorescence microscope (Zeiss Axio Observer, Zeiss Germany). Standardised recordings and uniform post-processing procedures were employed to ensure consistency. Six images per well were used to quantify the percentage area of live and dead cells in Fiji/ImageJ (https://imagej.net/ij/9) using the macro “measure stack” as described previously [29].

2.8 Enzyme-Linked Immunosorbent Assay (ELISA)

To measure the protein levels of human cytokines in the cell culture supernatant of each experimental group, the following ELISA kits were used: PDGF-AB/BB, VEGF and TGF-β1 (R&D Systems, Bio-Techne, Minneapolis, MN, USA). Cells were cultured without a complete change of media but fed with 200 μL of fresh medium on day 3. After 7 days, the medium was collected, centrifuged to separate cell debris and aliquoted for ELISAs. Simultaneously, small aliquots of each growth supplement, namely PRFe, PLP, and FBS, were collected on the day of preparation, on day 3 and on day 7. For each day, the supplements were pooled (n = 3) and stored at −80°C. Subsequently, we quantified the concentration of cytokines in the growth supplement using ELISA. The human protein levels were determined according to the manufacturer's instructions for each kit and optical density was measured using a plate reader (Infinite M Plex, TECAN, Männerdorf, Switzerland).

2.9 Multiplex qPCR

Cells were snap frozen in RNA Later (Qiagen, Hilden, Germany) and stored at −80°C. We used the TaqMan PreAmp Cells-to-CT Kit (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) and followed the manufacturer's recommended procedures for the preparation of our samples. After thawing, cells were washed in cold PBS and lysed using a 1:100 DNase 1 lysis solution. Following reverse transcription at 37°C for 60 min and 95°C for 5 min, the generated cDNA underwent preamplification (conditions: 95°C for 10 min, afterwards 10 cycles of 95°C for 15 s and 60°C for 4 min). Fluorescence-labelled probe-based multiplex RT-qPCR was performed with 5 μL of each cDNA sample and 15 μL of the TaqMan GenExpression Master Mix in a real-time thermocycler (qTowerG, Analytik Jena, Germany). We used the Eurofins qPCR-ASSAY software to design the primer sequences, which are listed in Table 2. The experiment was performed in biological triplicates (n = 3). The relative gene expression was normalised to the corresponding Housekeeping Index, consisting of the average of GAPDH. Afterwards, the transcript levels were calculated as 2^−ΔΔCt, in which ΔΔCt = ΔInduction—Δcontrol (basal medium).

2.10 Immunocytochemical Staining

Cells were seeded at a density of 2 × 10⁴ per well on cover glasses (Sarstedt AG & Co. KG Nuermbrecht, Germany) in 24-well plates (Greiner Bio-one, Frickenhausen, Germany) and treated with growth supplements according to our study design. After 24 h, 72 h and 7 days, the cell culture medium was discarded, and cells were washed with PBS twice. A 4% formaldehyde (Carl Roth, Karlsruhe, Germany) solution was used to fix cells for 20 min at room temperature and cells were then incubated in a blocking buffer containing PBS, 1% BSA and permeabilized by 0.03% Triton X-100 (Carl Roth, Karlsruhe, Germany) for 30 min. The cells were then washed twice with PBS and then incubated with primary antibodies TGF-beta1 (Human LAP (TGF-beta 1) Antibody, R&D Systems, Bio-Techne, Minneapolis, MN, USA) and CD44 (CD44 Monoclonal Antibody (IM7), Thermo Fisher Scientific, Waltham, MA, USA) for 1 h. Subsequently, the cells were washed again with PBS twice. Following the primary antibody incubation, appropriate secondary antibodies (IgG H&L) (Alexa Fluor 555, Abcam, Cambridge, UK) were added for 1 h.

To visualise the cell nuclei, DAPI (4′-6′-diamidino-2-phenylindole, Carl Roth, Karlsruhe, Germany) staining was performed by incubating cells for 5 min at room temperature.

2.11 Data Analysis

Each experiment was conducted in biological triplicates and technical duplicates. The statistical analysis was performed in GraphPad Prism (V10), with data tested for Gaussian distribution using a Shapiro–Wilk test. If the data followed a Gaussian distribution, statistical analysis was conducted using student's t-test or ANOVA for two or more variables. For significant results, a Bonferroni test was applied as post hoc analysis. Statistical significance was determined to be at least p < 0.05.

3.1 ASC Characterisation

Cells demonstrated uniform positive staining for the typical markers of ASCs such as CD13 (99.3% ± 0.9%), CD29 (97% ± 1.5%), CD44 (97.2% ± 2.3%), CD73 (99.4% ± 0.8%), CD90 (99.1.% ± 1.4%) and CD105 (87.8% ± 7.3%), while exhibiting homogeneous negative expression for primary negative markers including CD31 (1.8% ± 2.9%), CD45 (10.7% ± 5.7%) and CD235a (0.4% ± 0.3%). Moreover, these cells exhibited characteristics such as plastic adherence and self-renewal capability, thus meeting the classification criteria for ASC [26, 30]. Additionally, previous work from our department has elucidated the differentiation potential of the ASC we isolated as part of the characterisation [31].

3.2 Cell Viability and Proliferation

We studied the impact of three growth supplements on ASC-vitality in a 2D cell culture over a 7-day period. Generally, the metabolic activity measured in the AlamarBlue-Assay increased in all groups over time except for the basal medium only group. Moreover, the addition of PRFe significantly increased the cell viability of ASCs on day 7 when compared to other growth supplements (p < 0.05) (Figure 2A).

This finding is supported by the results of the Pico-Green Assay which showed a significant increase in DNA content in the PRFe group when compared to the other groups (p < 0.05 and p < 0.01) on day 7 (Figure 2B). Fluorescence staining of unfixed cells to evaluate the fraction of live and dead cells revealed that the PRF group had a higher number of vital cells when compared to the other groups (Figure 2C). Additionally, cell growth was analysed by calculation of covered well surface (Figure 2D). Here, PRFe showed significantly higher (p < 0.0001) covered well surface (90%) when compared to all other groups. Pure basal medium was inferior to all other groups.

3.3 Gene Expression

Compared to the 10% FBS group, a significant increase in the expression of JUN, COL1A1 (both p < 0.05) and NANOG (p < 0.01) was observed in the PRFe group after 7 days (Figure 3A–C). Exposure to PLP also resulted in an upregulation of these genes, although the increase was less pronounced and did not reach statistical significance. Likewise, treatment with 10% PRFe also led to an upregulation of SOX2 (twofold), PXN (1.5-fold), COX2 (twofold) and RPS6KA4 (3.6-fold) when compared to incubation with 10% FBS. However, due to the high standard deviation, these results were not statistically significant (data not shown).

3.4 Growth Factor Secretion

To identify cell signalling cascades involved in the observed effects of PRFe on ASC, we measured the concentration of several well-known cytokines known to be present in PRFe within the cell culture supernatant (Figure 4) as well as in the growth supplements of each corresponding experimental group (Figure 5). These included PDGF-AB, PDGF-BB, VEGF and TGF-β1 [3, 12]. After 7 days of culture, the concentration of TGF-ß1 was significantly higher (3- to 10-fold increase; p of at least < 0.05) in cells treated with PRFe when compared to the other groups (Figure 4A). ELISA of TGF-β1 in the growth supplements themselves over a period of 7 days indicated that the concentration was the highest in PRFe, even rising over time. PLP maintained a stable concentration of 2500–3000 pg/mL, whereas FBS showed a gradual decrease in TGF-β1 concentration over time with values under 100 pg/mL (Figure 5A).

VEGF secretion was the highest in the cell culture supernatant of cells treated with 10% PRFe compared to all other groups, although the difference did not reach statistical significance (Figure 4B). Analysis of growth supplements (PRFe, PLP and FBS) demonstrated VEGF to be present only in PRFe, with a peak concentration at day 3 (Figure 5B).

The concentration of PDGF-AB in the supernatant was elevated in all groups, ranging between 30 and 40 pg/mL, with a significantly higher concentration observed in the PRFe-group (Figure 4C). When comparing PDGF-AB levels in the growth supplements, it was found that PRFe had the highest concentration and showed a plateau after 3 days in culture (Figure 5C).

Like PDGF-AB, the concentration of PDGF-BB in the cell culture supernatant was significantly higher in cells treated with PRFe when compared to the other groups (p of at least < 0.05) (Figure 4D). Moreover, the analysis concentration of PDGF-BB in the growth supplements was highest in PRFe after three days of culture, with a slight decrease over time (Figure 5D).

3.5 Immunocytochemistry

After 24 h of incubation, ASC in all experimental groups expressed CD44 surface markers and demonstrated spindle-like morphology. Staining for intracellular TGF-beta1 was positive for 10% FBS and basal medium but only partially for 10% PLP and PRFe (Figure 6A).

Conversely, staining after 7 days revealed TGF-β1 in 10% PRFe. Moreover, these ASC expressed a higher density of CD44 and a more compact/smaller morphology (Figure 6B). As expected, cells exposed to basal medium without an additional growth supplement lost the ability to express CD44 and TGF-β1.