Cosmetic Medicine in Japan -東京大学美容外科- トレチノイン(レチノイン酸)療法、アンチエイジング(若返り)
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In cell-based regenerative therapies, transplantation of cells into target tissues usually takes place after cell manipulation, but most such manipulations (e.g., cell culture) require animal-derived products like serum or tissue extracts. Considering the risks, which include infection with viral or prion-related disease or immunological reactions,1 use of animal-derived products like fetal bovine serum (FBS) or bovine pituitary extract should be avoided. Thus, human-derived substances are considered the optimum materials for these manipulations. Autogolous serum obtained from whole blood enhances the expansion of human mesenchymal stem cells in culture.2-6 On the other hand, preparation for clinical use of component-level plasma products, such as platelet-rich plasma (PRP) or platelet-poor plasma (PPP), is considered to be less invasive because erythrocytes can be separately collected in the form of “high-density erythrocytes,” which can be given back to the patients.7,8
The proliferative effects in culture of various platelet derivatives like PRP, platelet-released supernatant, and platelet lysates on several human cell types have been reported.6,9-14 Our aim was to investigate the differences among three types of autologous serum?serum from whole blood (SWB), serum from PRP (SPRP), and serum from PPP (SPPP)?in their effects on the growth of three representative replicating human cells: human adipose-derived stem/stromal cells (hASCs), human dermal fibroblasts (hDFs), and human umbilical vein endothelial cells (HUVECs). It is well known that the bioactive protein levels secreted by platelets substantially differ among donors, platelet product types, processing techniques, and methods of platelet activation.15-21 Therefore, in this study, SWB, SPPP, and SPRP were prepared from the same four volunteers and evaluated for biochemical components and clinical potential as culture additives.

Materials and Methods
Collection and preparation of plasma and serum
Venous blood (300 mL each) was collected from four healthy volunteers after informed consent approved by our institutional review board (IRB). For preparation of PRP and PPP, the methods clinically applied for the preparations of autologous blood transfusion in our facility were used. The preparation protocol and blood components of whole blood (WB), PRP, and PPP are summarized in Figure 1A and B. Serum was prepared from WB, PRP, or PPP by elimination of coagulation factors such as fibrinogen, as described below.
First, 100 mL of each blood sample was drawn into a flask, and the remaining 200 mL of blood was drawn and stored in a blood bag (blood bag CPDA, Terumo, Tokyo, Japan) containing 0.327% citric acid, 2.63% sodium citrate, 0.0275% adenine, 0.251% sodium dihydrogen phosphate, and 2.9% D-glucose solution.
The blood in the flask was oscillated (agitated) at 37°C for one hour and incubated overnight at 4°C. The supernatant was collected using a 50-mL tube and centrifuged at 841 ?g for 10 minutes using a desktop centrifuge (KUBOTA 5200, Kubota, Co., Tokyo, Japan), and the supernatant was again collected as SWB. The stored 200 mL of blood in the blood bag was separated into 100 mL aliquots; one was centrifuged at 93 ?g for 10 minutes and the other at 841 ?g for 10 minutes. The resulting supernatants were PRP and PPP, respectively. The two types of plasma, PRP and PPP, were drawn into two flasks. After addition of 200 U of thrombin, the contents were oscillated (agitated) for 60 minutes at 37°C and incubated overnight at 4°C. The liquid component was drawn into a 50-mL tube and centrifuged at 841 ?g for 10 min, and the supernatants were obtained as SPRP and SPPP, respectively. The serum samples were frozen at -80°C and thawed at 37°C before analysis.

Biochemical analysis
A small portion of WB, PRP, and PPP was used for biochemical analysis to investigate the number of red blood cells (RBCs), white blood cells (WBCs), platelets, total protein (TP), albumin (Alb), sodium (Na+), potassium (K+), chloride (Cl-), and calcium (Ca++). Analysis was performed by SRL, Inc. (Tachikawa, Japan), a commercial analysis service.

Quantitative analysis of platelet-originated growth factors contained in serum
To analyze the concentration of platelet-originated growth factors in each serum sample, platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) were measured using respective anti-human ELISA kits (Quantikine, R&D Systems, Inc., MN) according to the manufacturer’s instructions. Levels of immunoreactive cytokines were measured at 450 nm by a microplate reader (Bio-Rad Laboratories Model 550, Hercules, CA), and a standard curve was generated to determine growth factor concentrations (pg/mL).

Preparation of human dermal fibroblasts (hDFs)
Human dermal fibroblasts (hDFs) were isolated from normal skin samples obtained from plastic surgery after informed consent approved by the IRB. The skin samples were cut into pieces of approximately 3 ? 3 mm in size and treated with 0.25% trypsin in phosphate buffered saline (PBS) solution for 24 hours at 4°C. After removal of the epidermis, the interstitial tissue fragments were attached to 100-mm plastic dishes, and cultured with DMEM (Nissui Pharmacertical, Tokyo, Japan) culture medium containing 10% FBS. Primary hDFs appeared in 4 to 7 days around the interstitial tissue fragments (after the initiation of outgrowth cultures) and became confluent after 2 to 3 weeks.

Preparation of human adipose-derived stem/stromal cells (hASCs)
Informed consent was obtained from each participant before collection of lipoaspirates from body-contouring surgery, according to the IRB-approved protocol. Human ASCs were isolated from the samples and cultured as previously described.22 In brief, the suctioned fat was digested with 0.075% collagenase in PBS solution for 30 minutes with agitation at 37°C. Mature adipocytes and connective tissues were separated from pellets by centrifugation at 800 ?g for 10 minutes. Cell pellets were passed through a 100-μm mesh filter (Millipore, MA) to remove debris and plated at a density of 5 ? 106 nucleated cells/100-mm plastic dish. Cells were cultured in M-199 medium containing 10% FBS at 37°C under 5% CO2 in a humidified incubator.

Preparation of human umbilical vein endothelial cells (HUVECs)
Before obtaining the placenta and umbilical cord samples, informed consent was obtained from each participant according to the protocol approved by IRB. Isolation and culture of HUVECs was done according to the method described by Jaffe et al.23 Samples were immediately collected after delivery, separating the umbilical cord from the placenta by clipping both ends, and irrigated using 1% iodine/PBS solution. To eliminate iodine, the intracelial space was rinsed using M-199 medium and filled with 0.25% trypsin in PBS. Both ends were again clipped, followed by incubation for 10 minutes at 37°C. Then, the intracelial space was rinsed using endothelial basal medium (EBM; Cambrex, Walkersville, MD), and cells were collected. The cells were centrifuged at 450 ?g for 5 minutes, attached to 100-mm plastic dishes, and cultured with EBM medium containing 2% FBS.

Cell proliferation assay using culture medium containing various sera
Standard culture media were prepared (DMEM for hDFs, M199 for hASCs, and EBM for HUVEC) containing FBS, SWB, SPRP, or SPPP at concentrations of 0%, 5%, and 10% for hDFs and hASCs, and of 0%, 1%, and 2% for HUVEC, respectively. A total of 5 ? 104 cells were plated in 60-mm dishes containing the prepared medium, and the medium was changed on the third and fifth days. Cells were counted on day 7 using a cell counter (NucleoCounter, Chemometec, Co., Allerod, Denmark). The average numbers were calculated from three different cultures for each cell type and culture condition.

Statistical analysis
Results were expressed as mean ± SE (standard error). To compare blood cell count and biochemical data for each sera, the values of SPRP and SPPP were described as a ratio to those of SWB. The Student’s unpaired t-test was used to evaluate the differences in influences on cell proliferation between FBS and human sera, while the Student’s paired t-test was used for different types of human sera. No correction was made for multiple comparisons. Statistical significance was defined as p < 0.05.

Blood cell count and biochemical analysis of WB, PRP, and PPP
The numbers of RBCs, WBCs, and platelets, and levels of total protein and albumin in WB at the time of blood collection were 428.0 (± 23.7) × 104 /μl, 6,100.0 ± 980.6 /μl, 25.0 (± 1.5) × 104 /μl, 7.0 ± 0.4 g/dl, and 4.2 ± 0.4 g/dl, respectively. As shown in Figure 2, in PRP, 75.1% of platelets remained in comparison with WB, although RBCs and WBCs decreased to 0.6% and 7.1%, respectively. In PPP, the number of platelets decreased to 12.6% with a decrement of RBCs and WBCs to 0.35% and 2.2%, respectively. Despite these reductions in blood cell numbers, the values for total protein and albumin were relatively uniform. In addition, we identified no notable changes in electrolyte levels.

PDGF and EGF concentrations in SWB, SPRP, and SPPP
Concentrations of PDGF and EGF were quantitatively analyzed by ELISA. As shown in Figure 3, in comparison with the values for SWB, SPRP contained 86.5% and 93.5% of PDGF and EGF respectively, while SPPP included only 19.1% and 11.2 % of those growth factors, respectively.

Effects of SWB, SPRP, and SPPP on the proliferation of various cell types
The number of proliferated cells in the culture media with different serum types at various concentrations was compared to that obtained by culture with FBS at the same concentration, using hDFs, hASCs, and HUVECs. Although the degree differed among the cell types, human-originated sera were effective for the expansion of cell number by culture.
In the hDF culture, SWB and SPRP exhibited a high proliferative efficacy that was almost identical to that of FBS, although cells cultured in SPPP showed a significantly lower degree of proliferation compared to other sera (Fig. 4A). Representative microscopic views of cultured hDFs are shown in Figure 4B.
In the hASC culture, although with the addition of SWB or SPRP cell proliferation outcome was inferior to that for FBS, the efficacy of cell proliferation was enhanced with increasing concentration of serum human products. There was no significant difference among effects of SWB, SPRP, and SPPP. Representative microscopic views of cultured hASCs are shown in Figure 5B.
Proliferation of HUVECs with SWB, SPRP, or SPPP did not significantly differ among the three types of human sera and was not as robust as that which occurred with addition of FBS (Fig. 6A). Representative microscopic views of cultured HUVECs are shown in Figure 6B.

Because specific gravity differs among various blood components, we can isolate each one by a specific centrifugation protocol; however, cell contamination cannot be completely avoided because of slight overlaps among these specific gravities. With our separation protocol, subtraction of RBCs and WBCs was sufficient in both PRP and PPP, and platelets were successfully preserved in PRP compared to PPP. Specifically regarding RBCs, less than 1% of the original numbers in WB remained in PRP or PPP. An expected advantage in the future use of these component-level serum (or plasma) products in regenerative medicine lies primarily in the possibility of salvage use of RBCs, conferring greater interest in SPRP and SPPP than in SWB.
Secretory proteins such as PDGF, EGF, transforming growth factor (TGF)-β1, and vascular endothelial growth factor (VEGF) are stored in the α-granules of platelets and released by platelet activation via addition of thrombin17,20 or adenosine diphosphate,20 or by a freeze/thaw cycle.11,16 Preparation and activation methods influence secretory protein concentrations.18,21 Platelet activation with thrombin, which we used in this study, is considered to closely imitate the physiologic activation of platelets, ensuring the bioactivity of secreted growth factors.6,24 In the present study, the concentration of total protein and albumin decreased slightly in the separation process of PRP and PPP from WB, but the concentrations of PDGF and EGF significantly decreased in proportion to the reduction in platelet count. Platelet-derived growth factors and platelet count were considered to be intimately associated, although the alteration was not linear.15,16,19
SWB and SPRP showed a high proliferative effect on hDFs, an effect almost identical to that of FBS, while hDFs cultured in SPPP showed a significantly lower degree of proliferation. Some platelet-originated growth factors, such as PDGF, are notable mitogens for hDFs.19,25 The difference in hDF proliferation effects among SWB, SPRP, and SPPP may primarily arise from differences in concentrations of the platelet-originated growth factors.
In ASC culture, although cell proliferation was generally enhanced depending on the concentrations of the three human serum preparations, cell proliferation outcome was inferior to that achieved with FBS. Our results using human sera obtained from the same four donors are inconsistent with the previous finding of Kocaoemer et al.6 that the proliferative efficacies of pooled human AB serum (corresponding to SWB in our study) and thrombin-activated PRP (corresponding to SPRP in our study) surpassed that of FBS. Platelets do not provide some major growth factors, such as basic fibroblast growth factor (b-FGF), keratinocyte growth factor, and hepatocyte growth factor.7,26 This may be the reason that human platelet-originated growth factors are not sufficient for expansion of hASCs; FBS may contain ingredients more influential for hASC proliferation, such as b-FGF.
For manipulating stem cells in regenerative medicine, differentiation capacity should be considered as well as proliferation capacity, and an optimal culture additive differs according to the purpose of the culture. Because PDGF is known to be a potent inhibitor of adipogenic differentiation of hASCs, SPPP with a selective addition of recombinant growth factors such as b-FGF and/or EGF may be preferable to SWB in hASC culture for adipose tissue engineering.14,27 In our study, differentiation capacity after cell expansion was not assessed because of the volume limitation of the samples.
In HUVEC culture, cell growth with either FBS or human serum preparations was inferior to that in a specific endothelial growth medium (data not shown), probably because growth factors such as b-FGF and VEGF that are not sufficiently present in serum are critical factors for HUVEC proliferation. The results of all human serum preparations were significantly worse than those obtained with FBS, although the three human serum preparations showed no significant differences among one another. A supplemental use of angiogenic growth factors may enhance the proliferative effect of serum products on HUVECs.
To our knowledge, this study is the first to compare different human serum preparations as an additive of cell culture, using blood samples obtained from identical donors. We found that SWB and SPRP are superior to SPPP as substitutes for animal-derived serum in culture expansion of hDFs. Platelet-derived ingredients, however, are considered non-essential or insufficient for enhanced proliferation of hASCs and HUVECs. Although autologous or human-derived serum preparations may be of great use in cell-based therapies in the future, this usefulness strongly depends on the target cell species and the purpose of the cell culture. Future studies should focus on establishing the optimal indications of each human serum preparation.

1. Tuschong, L., Soenen, S. L., Blaese, R. M., et al. Immune response to fetal calf serum by two adenosine deaminase-deficient patients after T cell gene therapy. Hum. Gene Ther. 13: 1605, 2002.

2. McAlinden, M. G., Wilson, D. J. Comparison of cancellous bone-derived cell proliferation in autologous human and fetal bovine serum. Cell Transplant. 9: 445, 2000.

3. Stute, N., Holtz, K., Bubenheim, M. et al. Autologous serum for isolation and expansion of human mesenchymal stem cells for clinical use. Exp. Hematol. 32: 1212, 2004.

4. Shahdadfar, A., Fronsdal, K., Haug, T. et al. In vitro expansion of human mesenchymal stem cells: choice of serum is a determinant of cell proliferation, differentiation, gene expression, and transcriptome stability. Stem Cells 23: 1357, 2005.

5. Anselme, K., Broux, O., Noel, B., et al. In vitro control of human bone marrow stromal cells for bone tissue engineering. Tissue Eng. 8: 941, 2002.

6. Kocaoemer, A., Kern, S., Kluter, H., et al. Human AB serum and thrombin-activated platelet-rich plasma are suitable alternatives to fetal calf serum for the expansion of mesenchymal stem cells from adipose tissue. Stem Cells 25: 1270, 2007.

7. Sanchez, A. R., Sheridan, P. J., and Kupp, L.I. Is platelet-rich plasma the perfect enhancement factor? A current review. Int. J. Oral Maxillofac. Implants. 18: 93, 2003.

8. Galel, S. A., Malone, III J. M., Viele, M. K. Transfusion medicine. In Wintrobe’s clinical hematology, 11th edition. Philadelphia: Lippincott Williams & Wilkins, 2004.

9. Lucarelli, E., Beccheroni, A., Donati, D., et al. Platelet-derived growth factors enhance proliferation of human stromal stem cells. Biomaterials 24: 3095, 2003.

10. Gruber, R., Karreth, F., Kandler, B., et al. Platelet-released supernatants increase migration and proliferation, and decrease osteogenic differentiation of bone marrow-derived mesenchymal progenitor cells under in vitro conditions. Platelets 15: 29, 2004.

11. Doucet, C., Ernou, I., Zhang, Y., et al. Platelet lysates promote mesenchymal stem cell expansion: a safety substitute for animal serum in cell-based therapy applications. J. Cell. Physiol. 205: 228, 2005.

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13. Vogel, J. P., Szalay, K., Geiger, F., et al. Platelet-rich plasma improves expansion of human mesenchymal stem cells and retains differentiation capacity and in vivo bone formation in calcium phosphate ceramics. Platelets 17: 462, 2006.

14. Koellensperger, E., von Heimburg, D., Markowicz, M., et al. Human serum from platelet-poor plasma for the culture of primary human preadipocytes. Stem Cells 24: 1218, 2006.

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Figure legends

Fig. 1. Preparation of three types of human serum
(A) For preparation of PRP or PPP, WB is separated by centrifugation into two components; PRP and the remainder (the RBC-containing part), or PPP and the remainder (the RBC-containing part). SPRP and SPPP were prepared through thrombin activation (blue triangle) of PRP and PPP, respectively.
(B) Blood components differ in specific gravity. Because of the differential specific gravity, PRP and PPP can be separated from WB by specific centrifugation protocols.
WB: whole blood, PRP: platelet-rich plasma, PPP: platelet-poor plasma, SWB: serum from WB, SPRP: serum from PRP, SPPP: serum from PPP, WBC: white blood cells, RBC: red blood cells.

Fig. 2. Blood counts and biochemical data in WB, PRP, and PPP
Values are expressed as ratios of biochemical parameters to those of WB. Student’s paired t-tests were used for statistical analysis. Black bars above indicate statistical significance (*p < 0.05, **p < 0.01). Values are mean, SE.
WB: whole blood, PRP: platelet-rich plasma, PPP: platelet-poor plasma, WBC: white blood cells, RBC: red blood cells, TP: total protein, Alb: albumin.

Fig. 3. Concentrations of platelet-derived cytokines in SWB, SPRP, and SPPP
Concentrations of two representative platelet-derived growth factors, EGF and PDGF, were measured in three types of serum prepared using various centrifugation conditions. Error bars indicate SE. Student’s paired t-tests were used for statistical analysis (*p < 0.05, ** p < 0.01).
SWB: serum from whole blood, SPRP: serum from platelet-rich plasma, SPPP: serum from platelet-poor plasma, EGF: epidermal growth factor, PDGF: platelet-derived growth factor.

Fig. 4. Human-dermal fibroblast (hDF) proliferation assay
(A) Cell counts of hDFs on day 7 of culture with addition of FBS, SWB, SPRP, or SPPP at concentrations of 0%, 5%, and 10%. Error bars indicate SE. Student’s paired t-tests were used for statistical analysis. Horizontal bars indicate statistical significance between different concentrations of each serum, or between different types of serum at the same concentration (*p < 0.05, **p < 0.01, ***p < 0.001).
(B) Representative microscopic views of hDFs (day 7) cultured with 10% FBS, SWB, SPRP, or SPPP.
FBS: fetal bovine serum, SWB: serum from whole blood, SPRP: serum from platelet-rich plasma, SPPP: serum from platelet-poor plasma.

Fig. 5 Human adipose-derived stem/stromal cell (hASC) proliferation assay
(A) Cell numbers of hASCs on day 7 cultured with addition of FBS, SWB, SPRP, or SPPP at concentrations of 0%, 5%, and 10%. Error bars indicate SE. Student’s t-tests were used for statistical analysis. Horizontal bars indicate statistical significance between different concentrations of each serum, or between different types of serum at the same concentration (*p < 0.05, **p < 0.01, ***p < 0.001).
(B) Representative microscopic views of hASCs (day 7) cultured with 10% FBS, SWB, SPRP, or SPPP.
FBS: fetal bovine serum, SWB: serum from whole blood, SPRP: serum from platelet-rich plasma, SPPP: serum from platelet-poor plasma.

Fig. 6. Human umbilical vein endothelial cell (HUVEC) proliferation assay
(A) Numbers of HUVECs on day 7 cultured with FBS, SWB, SPRP, or SPPP at concentrations of 0%, 1%, and 2%. Error bars indicate SE. Student’s paired t-tests were used for statistical analysis. Horizontal bars indicate statistical significance between different concentrations of each serum, or between different types of serum at the same concentration (*p < 0.05, **p < 0.01, ***p < 0.001).
(B) Representative microscopic views of HUVECs (day 7) cultured with 10% FBS, SWB, SPRP, or SPPP.
FBS: fetal bovine serum, SWB: serum from whole blood, SPRP: serum from platelet-rich plasma, SPPP: serum from platelet-poor plasma.

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