Cell and Tissue Banking

, Volume 9, Issue 1, pp 1–10 | Cite as

Human platelet lysate enhances the proliferative activity of cultured human fibroblast-like cells from different tissues

  • Vicente Mirabet
  • Pilar Solves
  • Mª Dolores Miñana
  • Araceli Encabo
  • Francisco Carbonell-Uberos
  • Amando Blanquer
  • Roberto Roig
Article

Abstract

Several studies have shown the presence of fibroblast-like cells in the stromal fraction of different tissues with a high proliferative and differentiation potential. Platelet alpha granules contain growth factors released into the environment during activation. The effects of different supplements for culture medium (human serum, bovine serum and platelet lysate) on cultured human fibroblast-like cells from bone marrow, adipose tissue, trabecular bone and dental pulp have been compared. Expression of typical stromal and hematopoietic markers was analyzed and proliferative rates were determined. Flow cytofluorometry showed a homogenous pattern in serial-passaged cells, with a high level of stromal cell-associated markers (CD13, CD90, CD105). The presence of platelet lysate in culture media increased the number of cell generations obtained regardless of cell source. This effect was serum-dependent. Cell-based therapies can benefit by the use of products from human origin for “ex vivo” expansion of multipotent cells.

Keywords

Platelet lysate Cell proliferation Human fibroblast-like cells Bone marrow Adipose tissue Trabecular bone Dental pulp Human serum 

Introduction

The presence of pluripotent and highly proliferative cells has been described in different human tissues: bone marrow (Hung et al. 2002; Pittenger et al. 1999; Reyes et al. 2001; Sekiya et al. 2002; Suva et al. 2004), adipose tissue (Gronthos et al. 2001; Halvorsen et al. 2001; Zuk et al. 2001; Planat-Benard et al. 2004), trabecular bone (Sakaguchi et al. 2004; Tuli et al. 2003), dental pulp (Gronthos et al. 2000; Miura et al. 2003), umbilical cord blood (Bieback et al. 2004; Erices et al. 2000; Lee et al. 2004) and peripheral blood (Zvaifler et al. 2000; Zhao et al. 2003; Yeh et al. 2003). In order to achieve sufficient amounts of these pluripotent cells for clinical purposes (Korbling and Estrov 2003) their ex vivo expansion is required (Horwitz et al. 2002; Le Blanc et al. 2004). Most culture models use fetal calf serum (FCS) as a supplement of culture media to increase the stem cell population in vitro. However, it has been determined that 7–30 mg of FCS proteins are associated with a standard preparation of 100 million human mesenchymal stem cells (Spees et al. 2004), and it may involve a humoral response (Horwitz et al. 2002). Moreover, these authors have shown that the efficiency of human serum could be improved with the use of epidermal growth factor (EGF) and basic fibroblast growth factor (FGF). Similarly, other authors have established a cell culture system for bone marrow stromal cells (based on human AB serum with the addition of FGF) as a feeder-layer for hematopoietic cell growth (Yamaguchi et al. 2002).

In human platelets there are granules containing cytokines and growth factors involved in tissue repair (Kaplan et al. 1979; Ledent et al. 1995). Some of the contents of these granules are: platelet-derived growth factor (PDGF), fibronectin, fibrinogen, transforming growth factor-β (TGF-β), insulin-like growth factor (IGF), epidermal growth factor (EGF), serotonin and fibroblast growth factor. There are different methods to obtain platelet-rich-plasma (PRP) for clinical use in regenerative medicine (Zimmermann et al. 2001; Wadhwa et al. 1996). In order to achieve degranulation, freezing and thawing to lyse cells and to release their content seems to be superior than inducing it by the addition of thrombin and calcium (Zimmermann et al. 2001).

Some authors have studied the effect of activated platelet concentrates on human primary cultures of fibroblasts and osteoblasts (Cenni et al. 2005), and others have studied this effect on cultured bone marrow stromal cells (Lucarelli et al. 2003). In these works platelet activation was achieved using thrombin to form a gel and fetal bovine serum was used to supplement culture media.

We describe the effect of platelet lysate supernatant (PLS) on cultured cells from different sources.

Materials and methods

Platelet processing

Platelets were obtained from routine plateletpheresis procedures in our regional transfusion center, using a blood cell separator (Haemonetics MSC+). This device was programmed to collect 3.3 ± 0.1 × 1011platelets per donor. Until processing, plasma-suspended platelets were stored at 22°C with continuous agitation. In order to assess storage conditions, the concentrations of PDGF-AB and TGF-β1 (both intracellular and released to supernatant) were evaluated by specific immunoassays according to the manufacturer’s instructions (R&D Systems). Standards and samples were assayed in duplicate, and mean values were calculated along several days of storage.

To achieve degranulation, platelet concentrates were centrifuged at 4,600g for 15 min. Then, plasma supernatant was discarded and the pellet was suspended in DMEM at a density of 4 × 109 plt/ml, immediately submerged in liquid nitrogen for 2 min and rapidly thawed in a water bath at 37°C. This freezing/thawing cycle was repeated twice. Finally, the suspension was centrifuged at 16,000g for 15 min to remove debris and the supernatant (PLS) was stored frozen at −30°C until use.

Cell cultures

Trabecular bone (n = 8) and bone marrow (n = 8) was obtained from the femoral heads of patients aged between 60 and 69 years undergoing hip arthroplasty. Human adipose tissue (n = 10) was obtained as liposuction aspirates from plastic surgery procedures from patients aged between 38 and 49 years. Dental pulp (n = 10) was obtained from normal molars freshly extracted for orthodontic reasons from young people (age 18–21 years).

Trabecular bone fragments from femoral heads were dissected into small pieces and digested with collagenase II (3 mg/ml) in Hank’s balanced salt solution (HBSS) at 37°C for 2 h with intermittent shaking. Bone marrow was obtained by aspiration from cancellous bone using a heparinized M199 solution. Adipose tissue was digested with collagenase I (1 mg/ml) in HBSS at room temperature with continuous shaking for 60 min. The teeth were immediately cracked opened, the pulp tissue was removed, minced into small fragments (<1 mm3) and then digested in a solution of collagenase type I (2 mg/ml) in HBSS for 90 min at 37°C, with intermittent shaking.

Digested tissue from bone and fat were filtered through a cotton surgical gauze. Cell suspensions were all centrifuged at 500 g for 10 min to collect the cells. Supernatants were discarded and cells suspended in 160 mM NH4Cl at room temperature for 10 min to lyse remaining red blood cells. Cells were collected by centrifugation as above and suspended in growth medium (GM: Dulbecco’s modified Eagle medium (DMEM) with low glucose, supplemented with 15% human serum and 25 μg/ml gentamicin. Then, primary cultures were seeded in culture flasks in a humidified atmosphere of 95% air and 5% CO2 at 37°C. This growth medium was refreshed every 2–3 days, removing nonadherent cells. When primary cultures became subconfluent, after 7–15 days, cells were collected by trypsinization (0.04% trypsin-0.02%EDTA solution in HBSS) and subcultured at a density of 5 × 10cells/cm2. First (P1) or second (P2) passage cells were used for proliferation assays with different culture media: (M1) GM; (M2) GM mixed (1:1) with conditioned medium (supernatant from fibroblastic cells cultured for 24 h with GM); (M3) GM with 5% PLS; (M4) GM with 10% PLS; (M5) GM mixed (1:1) with DMEM and supplemented with 10% PLS; (M6) DMEM with 10% PLS and gentamicine; and (M7) DMEM with 20% fetal bovine serum and gentamicine. Proliferation assays were performed in duplicate.

Culture yield was calculated counting in a Neubauer chamber. Population doubling time (PDT) was calculated with the formula PDT = log2(N/N0)/Tc where N0 and N were the inoculation cell count and the final cell count respectively, and Tc was the culture period, in hours.

PKH2 assays were performed with P1 or P2 cultures at subconfluence, according to manufacturer’s recommendations. The PKH2-GL (Sigma-Aldrich) cell linker kit incorporates a fluorescent dye with long aliphatic tails into lipid regions of the cell membrane. Cells were detached with trypsin/EDTA solution and centrifuged at 500g for 5 min. Supernatant was discarded leaving no more than 25 μl on the pellet. Then, 0.5 ml of diluent solution was added to suspend cells. Two milliliters of PKH2 solution was added to cell suspension and incubated for 5 min at room temperature. To stop reaction, 0.5 ml of human AB serum was added and incubated for 1 min. Finally, cell suspension was centrifuged (500 g for 5 min, 3 times) for washing and then suspended in culture medium. Five hundred thousand cells were fixed with paraformaldehyde (PFA) and stored as a pattern reference. From remaining cells, 103 cell/cm2 were cultured in two plates with different media (growth medium, M1; or PLS-supplemented growth medium, M4) until subconfluence. Then, they were detached and analyzed. PKH2 assays were performed in cultures from bone marrow (n = 2), adipose tissue (n = 2), trabecular bone (n = 3) and dental pulp (n = 3).

Flow cytometric analysis

To analyze cell-surface expression of typical markers, cultured cells were labeled with monoclonal antibodies against human antigens CD13-PE, CD34-APC, CD45-FITC, CD90-APC (all purchased from Becton Dickinson) and CD105-PE (Serotec). Fluorescein isothiocyanate (FITC), phycoerithrin (PE) and allophycocyanin (APC)-conjugated mouse IgG antibodies were used as isotype-matched controls. Actinomycin D (7-AAD) (Sigma-Aldrich) was used in order to exclude from the analysis non-viable cells making unspecific staining. Flow cytometric analysis was performed with a FACScalibur flow cytometer (Becton Dickinson) and CellQuest (Becton Dickinson) software. To analyze PKH2 assays ModFitLT v2.0 (Verity Software House) software was used.

Statistical analysis

Statistical Package for Social Sciences (SPSS v.10) was used to perform the statistical analysis. Means and standard deviations are shown for continuous variables. The Kolmogorov-Smirnov test was employed to investigate the normality distribution of the variables. As parameters were not distributed normally, statistical analysis was performed by non-parametric methods. The Mann–Whitney U test and Kruskal–Wallis test for continuous variables were used to compare the groups when applicable. We considered differences to be significant when P values were <0.05.

Results

The mean platelet count suspended in DMEM was 4,105 ± 190 × 10plt/ml. The concentration of intragranular PDGF-AB and TGF-β1 decreased until 70.6% and 92.9%, respectively, within 24 h of collection (Table 1). Thus, considering that we processed platelets immediately after collection, we could estimate the following concentrations in PLS: 272 ng/ml PDGF and 396 ng/ml TGF-β1.
Table 1

Intragranular load of PDGF-AB and TGF-β1 in platelet concentrates at collection and after 1, 3 and 5 days stored at 22°C with continuous shaking

 

PDGF-AB

TGF-β1

At collection

68 pg (100%)

99 pg (100%)

24 h

48 pg (70.6%)

92 pg (92.9%)

72 h

38 pg (55.9%)

78 pg (78.8%)

120 h

22 pg (32.3%)

42 pg (42.4%)

Concentrations are expressed as pg/10plt

The morphology of serially passaged cells was similar regardless of their source. Cells cultured in the presence of human serum were smaller and more spindle shaped than those cultured with bovine serum (Fig. 1). In cell populations from cultures supplemented with bovine serum, the percentage of small and agranular cells decreased to 35.9 ± 10.5% in relation to that observed in cultures with human serum. The proportion of these cells in primary cultures from different sources is shown in Table 2. The concentration of human serum influenced the presence of small cells in cultures from bone marrow. When human serum (HS) concentration was decreased from 20% to 10%, 5% and 2.5%, the percentage of these small cells similarly decreased to 80.5 ± 15%, 57.7 ± 19.9% and 50.6 ± 6.1%, respectively.
Fig. 1

Second passage cells from bone marrow cultured in medium supplemented with human serum (left) or bovine serum (right). The chart below corresponds to cell population distribution as a function of forward (size) and side (granularity) scattering. Two fractions can be distinguished: A, including small and agranular cells; and B, larger cells with different granularity degrees

Table 2

Percentage of small and agranular cells in primary culture cells from different tissues

Source

% Small cells

Bone marrow (n = 8)

8.8 ± 4.3

Trabecular bone (n = 8)

21.8 ± 6.7

Dental pulp (n = 10)

15.4 ± 5.5

Adipose tissue (n = 10)

8.8 ± 5.7

These cells were cultured with growth medium (containing human serum 15%, M1)

The immunophenotype of cultured cells is shown in Table 3. The most remarkable difference among primary cultures was the presence of a significant CD34 positive population in cells from adipose tissue. Nevertheless, the level of CD34 expression declined significantly with serial passage. Thus, second passage cells from all the tissues tested showed a homogeneous pattern with high level of stromal markers. The presence of PLS in culture media did not influence immunophenotype.
Table 3

Expression of stromal and hematopoietic cell-associated markers in primary culture cells (P0) and second passage cells (P2) from different sources (nd: non determined)

Source

CD45

CD90

CD105

CD34

CD13

P0

P2

P0

P2

P0

P2

P0

P2

P0

P2

Adipose tissue

±

+

++

±

++

++

±

+

+

Dental pulp

+

++

±

++

nd

nd

Trabecular bone

±

+

++

±

++

+

+

Bone marrow

±

+

++

±

++

++

++

The influence of media supplements is shown in Fig. 2. No differences were observed in growth kinetics comparing cultures fed with media supplemented with bovine (M7) or human serum (M1). The presence of PLS significantly reduced population doubling time (M3, M4 and M5) with respect to PLS-free medium (M1). However, similar results were obtained with growth medium regardless of PLS concentration (5%, M3; or 10%, M4) and even reducing human serum concentration to 7.5% but maintaining 10% PLS (M5). In addition, the effect of PLS on cultured cells was only evident when serum was present in culture medium. Figure 2 shows a decreasing pattern in population doubling time depending on cell source for all the assayed media (trabecular bone>bone marrow>adipose tissue>dental pulp).
Fig. 2

Graphic showing population doubling depending on cell source (bone marrow, adipose tissue, dental pulp or trabecular bone) and culture media (1–7, see Materials and methods to identify culture media composition). M6 (10% PLS in the absence of serum) proved to be inefficient to obtain cell growth from trabecular bone and dental pulp. First or second passage cells were used for these assays. *P < 0.05 **P < 0.005

With the use of PKH2 we can distribute cell populations as a function to the number of cell generations. Figure 3 shows population percentage in cell cultures fed with PLS10% (M4) or PLS-free medium (M1). This supplement significantly increased the percentage of cell populations undergoing a high number of cell generations for cultured cells from trabecular bone. Proliferation was also stimulated in cultured cells from bone marrow, but this effect was less evident with cells from dental pulp and even less with those from adipose tissue. Time to reach subconfluence was longer for trabecular bone and bone marrow cultures (6.2 ± 1.1 days) than those from dental pulp and adipose tissue (4.8 ± 0.4 days). Cell counts at subconfluence were similar regardless of cell source (data not shown).
Fig. 3

Influence of PLS on the number of cell generations in cultured cells (filled bars: growth medium; open bars: growth medium with 10% PLS). In all cases, a right deviation was observed in the number of cell generations when platelet factors were added to culture media. This effect was more evident in cultures from trabecular bone and less in those from adipose tissue

Discussion

To our knowledge, this is the first study simultaneously assessing the effect of platelet lysate supernatant on cultured cells from bone marrow, trabecular bone, dental pulp and adipose tissue. The use of cultured stromal cells from human adult tissues for cell-based therapies has a promising future for reconstituting damaged tissues. Nevertheless, the clinical use of FCS-cultured cells may lead to complications related to immune reactions (Spees et al. 2004; Horwitz et al. 2002; Hultman et al. 1996). Spees et al. have observed that the FCS proteins internalized by the cells were eliminated by further culturing with autologous serum and, furthermore, this process was optimized using conditions that promoted both metabolism and cell division (Spees et al. 2004). We propose a procedure that provides platelet-derived growth factors and cytokines promoting proliferation and metabolism. Simultaneously, the presence of human serum has been cited to contribute to smaller cells than those cultured with bovine serum (in bone marrow cells), then enriching the rapidly self-renewing cell fraction (RS) (Tuli et al. 2003) that has a high degree of clonogenicity and multipotentiality for differentiation (Sekiya et al. 2002; Colter et al. 2000; Mets and Verdonk 1981). Additionally, the use of growth factors has improved the proportion of RS cells in the cultures (Spees et al. 2004). We have observed a higher proportion of small and agranular cells (RS) with the use of human serum, but uninfluenced by PLS (data not shown). Our laboratory is part of a regional transfusion centre and thus human serum or platelet concentrates can be obtained from controlled donors, suitable for cell culture, at a very low cost. Results have not shown significant differences between cells cultured with human serum-supplemented medium (M1) or bovine serum-supplemented medium (M7), regardless of cell source.

To establish comparative studies among mesenchymal cell cultures is specially difficult for several reasons, mainly related to cellular heterogeneity. One of these reasons is the existence of variations in the number of colony forming units depending on tissue origin. Cells from adipose tissue mostly adhere to culture plastic surface and proliferate, but only a few cells attach in the case of cultures from bone marrow and trabecular bone. In fact, fibroblastic cell is a very scarce population in bone marrow and trabecular bone (Minguell et al. 2001; Friedenstein et al. 1982). Mitchell et al. have calculated the frequency of colony-forming unit fibroblasts in adipose tissue to be 1:32 (Mitchell et al. 2006). From our experience, in primary cultures from adipose tissue, 70–80% of seeded cells attached to plastic, most of them yielding colonies with fibroblast-like cells. Also related to cell suspension heterogeneity, the presence of hematopoietic nucleated cells in bone marrow and trabecular bone is higher than those in adipose tissue. Among these hematopoietic cells, there are cells which can adhere to plastic, limiting the available culture surface (e.g. monocytes) and/or releasing cytokines to supernatant (e.g. monocytes and granulocytes), then interfering the mesenchymal cell growth. Nevertheless, non-adherent blood cells are removed in supernatant with medium refreshing; and adherent blood cells remain adhered to culture surface when stromal fibroblastic-like cells are detached with trypsin/EDTA solution, because of their different sensibilities to the enzyme. Thus, hematopoietic cells are lost with culture passage. Hematopoietic cells are absent in cultures from dental pulp.

With regards to variability due to donor factors, Phinney et al. found differences in growth rates from bone marrow primary cultures up to 12-fold among donors, and without any correlation with donors’ age or gender. They proposed that it was due to cellular heterogeneity produced by the harvest method (Phinney et al. 1999). Variations have been described in cell suspensions, even when obtained from the same donor at the same time (DiGirolamo et al. 1999), and from the same tissue but from different locations (Prunet-Marcassus et al. 2006).

With regards to culture conditions, some authors have observed that initial cell density and time in culture affect cell morphology and colony forming unit efficiency (Sekiya et al. 2002). Other authors have suggested that mesenchymal stem cells enter senescence and start to lose their characteristics from the beginning of culture (Bonab et al. 2006).

Finally, there is no consensus as to the characteristic immunophenotype that can be used to distinguish passaged mesenchymal cells with different proliferation capabilities. Changes occur in expression of cell markers as a function of the culture (Mitchell et al. 2006; Pittenger et al. 1999; Suva et al. 2004). Nevertheless, as others, we have observed that serially passaged cells from different sources become homogeneous in phenotypic characterization for stromal cell markers.

The population doubling time (PDT) has been calculated considering the number of cells seeded and the number of cells harvested. However, this method is based on the assumptions that the cell population is homogeneous and that the first cell division after seeding is instantaneous. Some authors have observed that the time of the first cell division with cultured chondrocytes is highly variable (16–96 h) even among cells from the same donor and cultured in the same medium (Barbero et al. 2005). These authors also found that the presence of growth factors (PDGF-BB, TGFβ1 and FGF-2) in culture medium reduced the percentage of quiescent cells as well as the mean time for the first division and the generation time. Figure 3 shows that cultured cells become unsynchronized, containing populations showing proliferative heterogeneity. Cultures from bone marrow and trabecular bone yield more cells with a high number of cell generations but these cultures take longer to reach subconfluence that those from dental pulp and adipose tissue. These results suggest that growth in cultures from these last two tissues is sustained by a higher number of cells with proliferative potential than that observed in the other tissues. It is important to distinguish between PDT and doubling time (DT) (Hsieh and Graves 1998). The first is a property of the population and DT is a property of the cell (Deasy et al. 2003).

Platelet concentrates can be stored up to 5 days for clinical purposes. However, during this period, bioactive factors are released to supernatant as a consequence of spontaneous platelet activation (Zimmermann et al. 2001; Ledent et al. 1995). In order to avoid this leakage, we have processed platelets within the first 24 h from collection. In addition, to minimize the influence of other factors than platelet concentration on prediction to the final release of growth factors, the plateletpheresis procedure guaranteed the absence of white blood cells. Platelet-derived factors increase migration and proliferation, playing an important role in wound healing (Lowery et al. 1999; Robson 2003; Wiltfang et al. 2004). However, several studies indicate that factors released by platelets inhibit differentiation (Gruber et al. 2006; Kieswetter et al. 1997; Weiser et al. 1999) and it could be related to the application method used (pulse or continuous) (Hsieh and Graves 1998). Lucarelli et al. have demonstrated that 10% PRP is sufficient to induce a marked cell proliferation in stromal bone marrow cell cultures, even with serum-free medium (Lucarelli et al. 2003). Our results agree with this in the case of bone marrow and adipose tissue, but we have had difficulties to obtain cultures from trabecular bone and dental pulp using serum-free medium (M6) (Fig. 2). Cultured cells from these last two tissues remain attached and spreaded for several days but without signs of mitotic activity. We have observed massive cell detachment when human PLS was added to medium supplemented with bovine serum for 3T3 cells (cell line from murine fibroblast) growth (data not shown). The reason for this reaction to xenogeneic PLS is unknown for us. Monolayer cultures from fetal equine chondrocytes that were treated with TGF-β and supplemented with serum began to develop cellular toxicity (Nixon et al. 1998). Results with M6 seem to be related with an inefficacy to sustain cell growth from trabecular bone and dental pulp more than cellular toxicity. Probably, the higher percentage of small agranular cells observed in cultures from these tissues compared with those observed in cultures from bone marrow and adipose tissue (Table 2) could influence this fact. The increase observed in the proliferation rates from different cells using PLS in culture medium (M3, M4 and M5) has been significant compared to cultures fed with PLS-free growth medium (M1) (Fig. 2). In discordance with a study using platelet-rich plasma (Lucarelli et al. 2003) we have not observed a dose-dependent effect, but we have evaluated 5% and 10% concentrations of PLS whereas Lucarelli compared 1% and 10% concentrations of PRP.

First or second passage cells have been used in order to avoid the risk of cell transformation (Rubio et al. 2005) and to minimize changes due to aging. Other authors have shown that when platelet factors are withdrawn, cells return to their normal rate of proliferation (Lucarelli et al. 2003), which is relevant for safety reasons. From our experience, the presence of PLS has not influenced phenotype. Expression of cell markers have been similar in cell populations obtained with the use of growth medium (M1) or with PLS-supplemented growth medium (M4). No morphological changes have been observed in cultured cells when PLS was added to growth medium.

Our study supports the use of human serum for “ex vivo” expansion of human mesenchymal stem cells, avoiding the problems using bovine serum. We also show that PLS stimulates cell proliferation, increasing the number of cell generations in culture regardless of origin. Further comparative studies are needed to better understand not only the best method to expand human fibroblastic cells for therapeutic purposes, but also the most suitable source for these cells. Bone marrow is the most used tissue for this aim. However, in recent years, adipose tissue is emerging as a source of these cells. It is an accessible and abundant source that can be harvested with minimum patient discomfort, obtaining a higher yield in cells possessing comparable multipotency than those observed from bone marrow.

Notes

Acknowledgement

We wish to thank Isabel Plasencia, Casi Riol and Amparo Bernárdez for their excellent technical assistance.

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Copyright information

© Springer Science+Business Media B.V. 2007

Authors and Affiliations

  • Vicente Mirabet
    • 1
  • Pilar Solves
    • 1
  • Mª Dolores Miñana
    • 2
  • Araceli Encabo
    • 2
  • Francisco Carbonell-Uberos
    • 1
  • Amando Blanquer
    • 1
  • Roberto Roig
    • 1
  1. 1.Centro de Transfusión de la Comunidad ValencianaValenciaSpain
  2. 2.Fundación Hospital General Universitario de ValenciaValenciaSpain

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