CN109666635A - Platelet lysates liquid (HPL) substitutes method of fetal calf serum (FBS) culture for the bone marrow interstital stem cell of human body - Google Patents
Platelet lysates liquid (HPL) substitutes method of fetal calf serum (FBS) culture for the bone marrow interstital stem cell of human body Download PDFInfo
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- CN109666635A CN109666635A CN201710991038.3A CN201710991038A CN109666635A CN 109666635 A CN109666635 A CN 109666635A CN 201710991038 A CN201710991038 A CN 201710991038A CN 109666635 A CN109666635 A CN 109666635A
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Abstract
The present invention relates to can be avoided the negative effect that animal derived substance generates the clinical application of MSCs with HPL substitution FBS culture MSCs, cultured and amplified in vitro and clinical application to MSCs play corresponding directive function.HPL of the present invention can promote the skeletonization of MSCs/at rouge differentiation capability, from this, the MSCs of HPL culture may preferably be applied in bone tissue engineer field；Better curative effect may be obtained in the treatment and reparation of the skeletal tissue deficiencies such as osteogenesis imperfecta or damage disease, provides more efficient cell preparation for the cell therapy of these diseases.
Description
Technical field
The present invention relates to interstital stem cell and conversions.
Background technique
Interstital stem cell (Mesenchymal stem cells, MSCs), which is derived from mesoblastic one kind, has self more
New and multi-lineage potential stem cell, can isolate MSCs from the Various Tissues of multiple species, such as marrow, fatty group
It knits, peripheral blood etc..MSCs has multi-lineage potential, can be divided into osteoblast, fat cell, cartilage cell, cardiac muscle cell
Deng [1].Currently, MSCs is widely used in the clinical test treatment of field of tissue engineering technology and certain diseases,
Horwitz EM [2] etc. carries out allogene after receiving conventional bone-marrow transplantation to 6 children with osteogenesis imperfecta
MSCs infusion, the speed of growth of 5 infants is dramatically speeded up after MSCs is transfused, according to defeated to this 5 osteogenesis imperfecta infant MSCs
The observation of curative effect of note, author thinks the cell therapy that MSCs can be used for after osteogenesis imperfecta infant bone-marrow transplantation, and further mentions
MSCs may have certain curative effect to other heredity or acquired disease out.In addition, Quarto R etc. and Simon
Tilley etc. treats bone tissue defect using MSCs with Method of Tissue Engineering, obtains definite curative effect [3,4].
A large amount of MSCs is usually required when clinical application, it is now recognized that suitable clinical infusion dosage is adult per kilogram body
It is transfused 2 × 106 MSCs [19] again, therefore MSCs be used to have to pass through cultured and amplified in vitro when clinical treatment can be only achieved foot
Enough cell concentrations.MSCs is easy to in-vitro separation and amplification, and fetal calf serum (fetal bovine serum, FBS) is widely used
In the culture of MSCs, it is possible to the propagation for leading to prion or certain not yet diagnosed zoonosis, in addition, cultivating
The albumen or peptide of animal sources may be integrated into MSCs so as to cause immunological rejection in the process.The discovery such as Spees JL [5]
Rat produces apparent humoral immune reaction after being transfused the MSCs cultivated with FBS repeatedly；Horwitz EM [2] etc. is being utilized
Immune response is equally produced in the test of MSCs treatment children's osteogenesis imperfecta, has an infant to occur after being transfused MSCs twice
Nettle rash, and the infusion of MSCs to this infant without obvious curative effects.Therefore, some researchs are attempted to replace FBS with other substances
Cultivate MSCs, Bieback K [6] etc. replaces FBS to cultivate MSCs with human serum, blood plasma, but there is no good effect, blood
A large amount of hematopoietic cell can be mixed when clear or blood plasma culture MSCs, and MSCs growth rate in culture to forth generation obviously drops
It is low, it is extremely difficult to cell concentration required for clinical application；However employment platelet lysates liquid (human platelet lysate,
Disadvantages mentioned above is but not present when HPL) cultivating.HPL is to crack the blood plasma rich in growth factor obtained after blood platelet, wherein containing
By the main growth factor of secretion of platelet, such as platelet derived growth factor (PDGF), basic fibroblast growth factor
(b-FGF), transforming growth factor (TGF-β), insulin-like growth factor (IGF-I) etc., the culture of HPL can be higher than FBS
The promotion MSCs amplification in vitro of effect, while its multi-lineage potential and immunoloregulation function are kept, HPL can be used as FBS conjunction
Suitable and safety substitute.
Summary of the invention
Technical problem to be solved by the present invention lies in provide a kind of platelet lysates liquid (HPL) substitution fetal calf serum
(FBS) method of the culture for the bone marrow interstital stem cell of human body.
The present invention solves its technical problem, the technical solution adopted is that, a kind of platelet lysates liquid (HPL) substitution tire is provided
Method of cow's serum (FBS) culture for the bone marrow interstital stem cell of human body, which is characterized in that collect 10 bags of Single-donor platelets
(0.2 unit of each donor) freezes at -80 DEG C, then quickly dissolves at 37 DEG C.900r/min is centrifuged 30min.Supernatant is taken,
0.22 μm of filter filtering, adds heparin 2U/ml to prevent the formation of platelet gel.Platelet lysates liquid uses ELISA method
It carries out PDGF-AB (vectors containing human platelet-derived growth AB), (Insulin-Like is raw by bFGF (fibroblast growth factor), IGF-1
The long factor 1), the detection of TGF-β 1 (transforminggrowthfactor-β1) 4 kinds of cytokine concentrations
Preferably, during 7.5%HPL and 10%FCS culture MSCs is primary, occur in inoculation 5d or so adherent thin
Born of the same parents are in spindle shape, and cloning is proliferated, and 10~15d reaches fusion, had digestive transfer culture.
Preferably, MSCs was cultivated to the 5th generation, was digested, and surface markers CD45, CD34, CD29, CD44 are detected,
CD105, CD106.
Preferably, its cell cycle of MSCs of 10% FCS culture is the S phase: (1.42 ± 1.83) %, G0-G1 phase:
(96.97 ± 0.95) %, G2-M phase: (1.61 ± 1.56) %；Its cell cycle of MSCs of 7.5% HPL culture is the S phase
(3.54 ± 2.55) %, G0-G1 phase: (94.58 ± 1.56) %, G2-M phase: (1.88 ± 1.64) %；It can from above-mentioned data
For G0~G1 phase cell proportion 90% or more, this illustrates that most cells are in resting state out, retain self-renewing and
Proliferative capacity, this meets the characteristic of stem cell
Preferably, osteogenic induction is carried out to culture to MSCs under HPL the and FCS condition of culture in the 5th generation respectively, induces 10-
The tubercle of visible densification agglomerate sample is formed under 12 days or so light microscopics, and tubercle differs in size, and Alizarin red staining, tubercle quilt are carried out to it
Dye is red, it was demonstrated that is calcium tubercle
Preferably, adipogenic induction equally is carried out to culture to two groups of MSCs under the 5th generation HPL and FCS condition of culture, lured
When leading 8 days in most cells fat drips, it is apparent that with induction time extension, small fat drips are gradually gathered into big rouge
Bubble is dyed to red with fat drips in oil red O stain visible cell after induction 12 days.
The beneficial effects of the present invention are: HPL of the present invention can promote the skeletonization of MSCs/at rouge differentiation capability, from this,
The MSCs of HPL culture may preferably be applied in bone tissue engineer field；In the skeletal tissue deficiencies such as osteogenesis imperfecta or damage
Better curative effect may be obtained in the treatment and reparation of disease, provides more efficient cell system for the cell therapy of these diseases
Agent.
Specific embodiment
It can be avoided the negative shadow that animal derived substance generates the clinical application of MSCs with HPL substitution FBS culture MSCs
It rings, cultured and amplified in vitro and clinical application to MSCs play corresponding directive function.This patent HPL can promote MSCs at
Bone/and at rouge differentiation capability, from this, the MSCs of HPL culture may preferably be applied in bone tissue engineer field；At
Better curative effect may be obtained in the treatment and reparation of the not congruent skeletal tissue deficiencies of bone or damage disease, is the cell of these diseases
It treats and more efficient cell preparation is provided.
It collects 10 bags of Single-donor platelets (0.2 unit of each donor) to freeze at -80 DEG C, then quickly be dissolved at 37 DEG C.
900r/min is centrifuged 30min.Supernatant is taken, 0.22 μm of filter filtering adds heparin 2U/ml to prevent the formation of platelet gel.
Platelet lysates liquid carries out PDGF-AB (vectors containing human platelet-derived growth AB) using ELISA method, bFGF (fibroblast
Growth factor), IGF-1 (type-1 insulin like growth factor), the inspection of TGF-β 1 (transforminggrowthfactor-β1) 4 kinds of cytokine concentrations
It surveys.
1.MSCs is proliferated growing state in HPL and FCS
During 7.5%HPL and 10%FCS culture MSCs is primary, there is attached cell in inoculation 5d or so, be in long shuttle
Shape, cloning proliferation, 10~15d reach fusion, had digestive transfer culture.The MSCs of HPL culture is opposite
Cluster growth is tended to for FCS, and the time of the MSCs pancreatin digestion of HPL culture is 2~3min, and FCS
The time of the MSCs pancreatin digestion of culture is 3~5min.It is in spindle shape that 10%FCS, which cultivates cell, and per generation 3d reaches fusion,
7.5%HPL culture cell shape is similar to 10%FCS culture cell, is also spindle shape, per generation 4d reaches fusion.Take 6 plants of cells
P5OD value carries out the t- between two groups independent and examines, P > 0.05, therefore 7.5%HPL culture ability of cell proliferation and 10%FCS
There is no significant difference between culture ability of cell proliferation.
The identification of 2.HPL and FCS condition of culture lower surface label
MSCs was cultivated to the 5th generation, was digested, and surface markers CD45, CD34, CD29, CD44, CD105, CD106 are detected.
As the result is shown compared with the MSCs of FCS culture, HPL cultivates the expression (P > 0.05) for its surface markers that do not have an impact.
The identification of 3.HPL and FCS condition of culture lower cell cycle
Its cell cycle of MSCs of 10% FCS culture is the S phase: (1.42 ± 1.83) %, G0-G1 phase: (96.97 ±
0.95) %, the G2-M phase: (1.61 ± 1.56) %；7.5% HPL culture its cell cycle of MSCs be the S phase (3.54 ±
2.55) %, the G0-G1 phase: (94.58 ± 1.56) %, G2-M phase: (1.88 ± 1.64) %；From above-mentioned data can be seen that G0~
For G1 phase cell proportion 90% or more, this illustrates that most cells are in resting state, retains self-renewing and proliferation energy
Power, this meets the characteristic of stem cell.The MSCs cell cycle under two kinds of system condition of culture is examined by t, and P > 0.05 shows
There is no significant differences in terms of the MSCs cell cycle for two kinds of cultivating systems.
4. Osteoinductive differentiation and Quantitative measurement result
Osteogenic induction is carried out to culture to MSCs under HPL the and FCS condition of culture in the 5th generation respectively, is induced 10-12 days or so
The tubercle of visible densification agglomerate sample is formed under light microscopic, and tubercle differs in size, and Alizarin red staining is carried out to it, it is red that tubercle, which is contaminated,
Color, it was demonstrated that be calcium tubercle.Quantitative measurement and statistical analysis are carried out to the Osteoblast Differentiation result of two groups of MSCs of three different samples,
The MSCs skeletonization quantitative result of L-DMEM culture containing 10%FBS is 4.151 ± 5.631, and the training of the L-DMEM containing 7.5%HPL
Feeding MSCs skeletonization quantitative result is 16.548 ± 4.397, and the skeletonization quantitative result discovery HPL for comparing two groups of MSCs can significantly promote
Into the Osteoblast Differentiation ability of MSCs, P < 0.05.
5. adipogenic induction differentiation and Quantitative measurement result
Adipogenic induction equally is carried out to culture to two groups of MSCs under the 5th generation HPL and FCS condition of culture, when inducing 8 days
In most cells fat drips, it is apparent that with induction time extension, small fat drips are gradually gathered into big fat vacuole, induce
Red is dyed to fat drips in oil red O stain visible cell after 12 days.To two groups of MSCs of three different samples at rouge knot
Fruit carries out Quantitative measurement and statistical analysis, the MSCs of the L-DMEM culture containing 10%FBS at rouge quantitative result be 0.497 ±
0.105, and the MSCs of the culture of the L-DMEM containing 7.5%HPL is 0.239 ± 0.030 at rouge quantitative result, compares two groups of MSCs'
Inhibit MSCs to Adipocyte Differentiation (P < 0.05) at rouge quantitative result discovery HPL.
It should be understood that above embodiments only express the preferred embodiment of the present invention, description is more specific and detailed
Carefully, but it cannot be understood as limitations on the scope of the patent of the present invention；It should be pointed out that for the common skill of this field
For art personnel, without departing from the inventive concept of the premise, above-mentioned technical characterstic can be freely combined, can also be done
Several modifications and improvements out, these are all within the scope of protection of the present invention；Therefore, all to be done with scope of the invention as claimed
Equivalents and modification, should belong to the covering scope of the claims in the present invention.
Claims (6)
1. a kind of side of bone marrow interstital stem cell of platelet lysates liquid (HPL) substitution fetal calf serum (FBS) culture for human body
Method, which is characterized in that collect 10 bags of Single-donor platelets (0.2 unit of each donor) and frozen at -80 DEG C, then quickly at 37 DEG C
Dissolution.900r/min is centrifuged 30min.Supernatant is taken, 0.22 μm of filter filtering adds heparin 2U/ml to prevent platelet gel
It is formed.Platelet lysates liquid carries out PDGF-AB (vectors containing human platelet-derived growth AB) using ELISA method, and bFGF is (at fiber
Porcine HGF), IGF-1 (type-1 insulin like growth factor), 4 kinds of cytokine concentrations of TGF-β 1 (transforminggrowthfactor-β1)
Detection.
2. the method according to claim 1, wherein 7.5%HPL and 10%FCS culture MSCs it is primary during,
There is attached cell in inoculation 5d or so, be in spindle shape, cloning is proliferated, and 10~15d reaches fusion, had digestive transfer culture.
3. being digested the method according to claim 1, wherein MSCs was cultivated to the 5th generation, detection surface mark
Remember CD45, CD34, CD29, CD44, CD105, CD106.
4. the method according to claim 1, wherein its cell cycle of MSCs that 10% FCS is cultivated is the S phase:
(1.42 ± 1.83) %, G0-G1 phase: (96.97 ± 0.95) %, G2-M phase: (1.61 ± 1.56) %；7.5% HPL culture
Its cell cycle of MSCs is S phase (3.54 ± 2.55) %, the G0-G1 phase: (94.58 ± 1.56) %, G2-M phase: (1.88 ±
1.64) %；It can be seen that G0~G1 phase cell proportion 90% or more from above-mentioned data, this illustrates at most cells
In resting state, retain self-renewing and proliferative capacity, this meets the characteristic of stem cell.
5. the method according to claim 1, wherein under culture to the HPL and FCS condition of culture in the 5th generation
MSCs carries out osteogenic induction respectively, induces the tubercle of visible fine and close agglomerate sample under 10-12 days or so light microscopics to be formed, tubercle size is not
Deng carrying out Alizarin red staining to it, tubercle is contaminated for red, it was demonstrated that be calcium tubercle.
6. the method according to claim 1, wherein equally under culture to the 5th generation HPL and FCS condition of culture
Two groups of MSCs carry out adipogenic induction, induce 8 days when most cells in fat drips, it is apparent that with induction time
Extend, small fat drips are gradually gathered into big fat vacuole, are dyed to after induction 12 days with fat drips in the dyeing visible cell of oil red 0 red
Color.
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| CN201710991038.3A CN109666635A (en) | 2017-10-16 | 2017-10-16 | Platelet lysates liquid (HPL) substitutes method of fetal calf serum (FBS) culture for the bone marrow interstital stem cell of human body |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114032209A (en) * | 2021-11-23 | 2022-02-11 | 广东普罗凯融生物医药科技有限公司 | Kit for separating dental pulp stem cells and application thereof |
| CN115521906A (en) * | 2021-06-24 | 2022-12-27 | 东莞宣冠干细胞再生医学有限公司 | Method for preparing high-quality human-derived dental pulp stem cells |
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- 2017-10-16 CN CN201710991038.3A patent/CN109666635A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115521906A (en) * | 2021-06-24 | 2022-12-27 | 东莞宣冠干细胞再生医学有限公司 | Method for preparing high-quality human-derived dental pulp stem cells |
| CN114032209A (en) * | 2021-11-23 | 2022-02-11 | 广东普罗凯融生物医药科技有限公司 | Kit for separating dental pulp stem cells and application thereof |
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