Cell Technologies in Biology and Medicine, No. 3, November, 2013
146
Human Platelet Lysate as a Promising Growth-Stimulating Additive for Culturing of Stem Cells and other Cell Types
Ya. D. Shanskii1, N. S. Sergeeva1,2, I. K. Sviridova1, M. S. Kirakozov2, V. A. Kirsanova1, S. A. Akhmedova1, A. I. Antokhin2, and V. I. Chissov1,3 Translated from Kletochnye Tekhnologii v Biologii i Meditsine, No. 3, pp. 153-158, July, 2013 Original article submitted January 14, 2013 We compared the composition and biological activity of fetal calf serum and platelet lysate from donor platelet concentrate. In platelet lysate, the concentrations of alkaline phosphatase, lactate dehydrogenase, creatinine, and mineral metabolism parameters were lower, while parameters of lipid and protein metabolism were higher than in fetal calf serum. The concentrations of growth factors (platelet-derived (AA, AB, BB), vascular endothelial, insulin-like, and transforming growth factor β) in platelet lysate 1.7-148.7-fold surpassed the corresponding parameters in fetal calf serum. After replacement of fetal calf serum with platelet lysate in the culture medium (0, 25, 50, 75, and 100%), the count of multipotent mesenchymal stromal cells on day 7 (in comparison with day 1) increased by 154.8, 206.6, 228.2, 367.7, and 396.5%, respectively. Thus, platelet lysate can be an adequate non-xenogenic alternative for fetal calf serum. Key Words: human platelet lysate; fetal calf serum; multipotent mesenchymal stromal cells; cell culture The progress in cell techniques and tissue engineering requires the development of safe technologies for culturing of cells intended for administration to patients, in particular involving the use of safe culture media. Fetal calf serum (FCS) is an essential component of culture media for various cell types; it is the source of various growth factors providing vital activity and proliferation of cells in culture [3]. However, the use of xenogenic additives for manufacturing cell products intended for administration to patients are prohibited in the majority industrial countries. This is related to possible transfer, in particular from FCS, of infectious agents, including non-identified ones (prions, zoonotic pathogens, etc.) and the risk of allergic and anaphylactic reactions, including graft-versushost reaction [3,4]. 1
P. A. Gertsen Moscow Research Oncological Institute; 2N. I. Pirogov Russian National Research Medical University; 3I. M. Sechenov First Moscow State Medical University, Ministry of Health Care of the Russian Federation, Moscow, Russia. Address for correspondence:
[email protected]. D. D. Shanskii
Moreover, high lot-to-lot variability of FCS composition hampers standardization by the most relevant components and creates difficulties in selecting FCS for certain cell lines (e.g. multipotent mesenchymal stromal cells, MMSC) and sometimes in comparing the results obtained with the use of different lots of FCS. These circumstances hamper standardization and registration of cell products [3,6]. Numerous attempts of cell culturing in serum-free media were undertaken to solve the problems related to the use of FCS for manufacturing cell products and their derivatives. However, the cells can change the properties and phenotype under non-physiological conditions, while artificial serum-free media maintaining initial cell properties have not yet been created [4]. Much recent attention was attracted to platelets as a possible source of growth factors. Platelets are traditionally used in medical practice for the therapy of conditions associated with massive blood loss and blood clotting disturbances. Coagulation activity of platelets is determined by the presence thrombospon-
0007-4888/13/15610146 © 2013 Springer Science+Business Media New York
Ya. D. Shanskii, N. S.Sergeeva, et al.
147
din, fibrinogen, fibronectin, etc. Enzyme immunoassay has shown that the thrombus is the source of not only clotting factors, but also growth factors. It has been demonstrated that platelets contain a set of growth factors that are released into the blood during their lysis: platelet-derived growth factor (PDGF and its isoforms AA, AB и BB), fibroblast growth factor (FGF), tissue growth factor в (TGF-β), EGF, vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), etc. [3,5]. Platelet concentration in commercially available platelet preparation, platelet concentrate, 4-fold surpasses that in normal human plasma. Studies of platelet lysate (PL) prepared from human platelet concentrate as the source of growth factors for cell culturing were started few years ago. For instance, an increase in the number of human fibroblast CFU during culturing with PL and the possibility of culturing MMSC in the presence of PL were reported [6-8].
Here we determined quantitative biochemical parameters and concentrations of growth factors in FCS and human PL and evaluated their effects as medium supplements on the growth of MMSC in culture.
MATERIALS AND METHODS PL isolation. Platelet concentrate obtained from 27 male and 19 female donors (mean age 33 years: males 31 years and women 35 years) was used for PL preparation. The majority of donors were under 45. PL was prepared by thermal lysis accompanied by degradation of α-granules and release of growth factors. Platelet concentrate from each donor was transported from the Department of Blood Transfusion, P. A. Gertsen Moscow Research Oncological Institute, in 5-6-ml sample tubes. The tubes with the platelet concentrate were frozen at -80oC and then rapidly defrosted at 37oC (thermal lysis). Then, the content of
TABLE 1. Biochemical Parameters of FCS and PL Parameter
NR
FCS (M±m; n=4)
PL (M±m; n=20)
0-20.5
1.18±0.33
3.16±0.32
Cholesterol, mmol/liter
3.12-6.24
0.88±0.08
3.83±0.17*
HDL, mmol/liter
0.91-1.56
0.17±0.02
1.14±0.04*
LDL, mmol/liter
0-3.36
0.42±0.07
1.72±0.12
Triglycerides, mmol/liter
0-2.26
0.90±0.15
1.03±0.13
Total protein, g/liter
64-83
36.50±1.24
58.64±0.77*
Albumin, g/liter
35-50
20.98±1.36
34.45±0.45*
AST, U/liter
0-40
18.67±8.88
39.20±2.17
ALT, U/liter
0-41
3.75±0.25
17.20±1.50
Alkaline phosphatase, U/liter
0-270
678.00±37.23
112.85±6.70*
LDH, U/liter
225-450
702.25±70.89
379.55±61.00*
Urea, mmol/liter
2.5-8.3
6.10±0.17
4.56±0.22
Uric acid, μmol/liter
137-452
68.25±14.33
246.60±15.89*
Glucose, mmol/liter
3.3-6.1
3.53±0.83
15.94±0.23*
Potassium, mmol/liter
3.5-5.0
11.90±0.55
4.79±0.09*
Sodium, mmol/liter
135-152
140.18±0.77
157.13±0.62*
Calcium, mmol/liter
2.15-2.58
3.44±0.10
2.02±0.02*
Inorganic phosphorus, mmol/liter
0.81-1.45
3.20±0.28
1.35±0.17*
96-108
98.50±0.87
85.35±0.50*
Magnesium, mmol/liter
0.70-1.05
1.42±0.06
0.88±0.01*
Iron, μmol/liter
9.0-31.3
32.68±5.14
14.79±0.98*
53-115
206.75±14.51
84.35±1.69*
Total bilirubin, μmol/liter
Chlorides, mmol/liter
Creatinine, μmol/liter
Note. Here and in Table 2: NR: normal range for the parameter in human venous blood. HDL: high-density lipoproteins; LDL: low-density lipoproteins; n: number of samples. Parameters beyond the normal range for human venous blood are displayed in bold type. *p
