CN108795857A - A kind of cell culture fluid prepares the method for platelet rich plasma and platelet rich plasma obtained using the cell culture fluid - Google Patents
A kind of cell culture fluid prepares the method for platelet rich plasma and platelet rich plasma obtained using the cell culture fluid Download PDFInfo
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- CN108795857A CN108795857A CN201810638692.0A CN201810638692A CN108795857A CN 108795857 A CN108795857 A CN 108795857A CN 201810638692 A CN201810638692 A CN 201810638692A CN 108795857 A CN108795857 A CN 108795857A
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Abstract
The invention belongs to biomedicine technical fields, disclose a kind of cell culture fluid, are mainly grouped as by the group of following volumes percentage：2.5% vitamin C, 2.5% N- acetylamino acids, 1.5% branched-chain amino acid, surplus are conventional cell culture medium.The present invention also provides the methods for extracting thrombocyte plasma using the cell culture fluid.Cell culture formula of liquid provided by the invention is simple, at low cost, can effectively facilitate blood platelet and the leucocyte secretion beneficial growth factor in blood.The cell culture fluid is combined with the preparation method of platelet rich plasma provided by the invention, can quickly prepare containing a large amount of beneficial growth factors and it is pollution-free can direct injection platelet rich plasma, can effectively treat alopecia seborrheica.
Description
Technical field
The invention belongs to biomedicine technical fields, and in particular to a kind of cell culture fluid utilizes the cell culture fluid system
The method of standby platelet rich plasma and platelet rich plasma obtained.
Background technology
The patient of alopecia seborrheica is often due to androgen metabolic disorder in local organization, passes through the effect of 5 alpha-reductases
The testosterone for keeping activity low is converted into the high dihydrotestosterone (DHT) of activity, and DHT has very strong affinity with androgen receptor, can
It works in conjunction with and into nucleus with androgen receptor, hair follicle precocious, growth period is made to shorten and enter stand-down in advance,
Hair follicle gradual change is small, and hair becomes thin short, and then alopecia, hair transplant is usually used for the treatment of alopecia at present, there are no effective
Means can make hair loss patient hair tonic in a short time.Platelet rich plasma (PRP) is to be rich in blood using what self-blood made
The high-concentration blood plasma of platelet, wherein including a variety of growth factors such as vascular endothelial growth factor (VEGF), transforming growth factor
TGF-β 1, platelet derived growth factor (PDGF), insulin-like growth factor I GF-I, IGF-II, fibrin, fiber knot
The factor of hop protein molecule, promotion inflammatory reaction, cell Proliferation and differentiation etc., using relatively wide in surface of a wound injury repair.
However, the extraction of PRP is mainly secondary centrifuging method at present, have the following disadvantages：Open preparation system
It is easy, by outside contamination, to shift between multiple containers when extracting PRP, increases the probability that blood platelet is contaminated and activates, behaviour
The personal operating habit for making personnel greatly affected the concentration of blood platelet in PRP, and factors above causes blood in the PRP prepared small
Plate acquisition rate is relatively low and each index coefficient of variation is big, dramatically limits the application of PRP.Therefore, it is badly in need of developing a kind of energy
Rapid extraction contains a large amount of beneficiating ingredients and the method for PRP not easy to pollute.
Invention content
In order to solve the above problem of the existing technology, present invention aims at a kind of cell culture fluid of offer, utilizations
The cell culture fluid prepares the method for platelet rich plasma and platelet rich plasma obtained.
The technical solution adopted in the present invention is：
The present invention provides a kind of cell culture fluids, are mainly grouped as by the group of following volumes percentage：2.5% dimension life
Plain C, 2.5% N- acetylamino acids, 1.5% branched-chain amino acid, surplus are conventional cell culture medium.
Specifically, above-mentioned cell culture fluid, amino acid L-methionine, L- threonines, L- in the N- acetylamino acids
Histidine, L-Glutamine, l-Isoleucine, L-Leu, L-lysine, L-phenylalanine, L-Trp or Valine,
The branched-chain amino acid is leucine.
Specifically, above-mentioned cell culture fluid, the cell culture medium be DMEM culture mediums, H3 serum free mediums or
RPMI-1640 culture mediums.
The present invention also provides a kind of preparation methods of platelet rich plasma, include the following steps：
S1：Peripheral blood is taken, cell culture fluid above-mentioned is added into peripheral blood, mixing obtains mixed liquor one；
S2：By one stationary culture of above-mentioned mixed liquor, mixed liquor two is obtained；
S3：Mixed liquor two described in step S2 is centrifuged, takes supernatant and intermediate cell layer to get to the rich platelet
Blood plasma.
Specifically, above-mentioned preparation method, the volume of peripheral blood and the ratio of the cell culture fluid are in the step S1
20:1。
Specifically, above-mentioned preparation method, the temperature cultivated in the step S2 is 37 DEG C, CO when culture2It is a concentration of
5%, incubation time 4h.
Specifically, above-mentioned preparation method, centrifugal force is 26G, centrifugation time 15min when being centrifuged in the step S3.
A kind of platelet rich plasma prepared by aforementioned preparation process.
Beneficial effects of the present invention are：
Cell culture formula of liquid provided by the invention is simple, at low cost, can effectively facilitate blood platelet in blood and white thin
The intracrine beneficial growth factor.Using cell culture fluid provided by the invention and combine platelet rich plasma provided by the invention
Preparation method, can quickly prepare containing a large amount of beneficial growth factors and it is pollution-free can direct injection platelet rich plasma, should
Platelet rich plasma can effectively treat alopecia seborrheica.
Description of the drawings
Fig. 1 is the design sketch of platelet rich plasma treatment severe seborrheic alopecia provided by the invention；
Fig. 2 is the design sketch that platelet rich plasma provided by the invention treats common alopecia seborrheica.
Specific implementation mode
Below in conjunction with the accompanying drawings and specific embodiment does further explaination to the present invention.
Embodiment 1
The purpose of the present embodiment is that providing a kind of cell culture fluid, mainly it is grouped as by the group of following volumes percentage：
2.5% vitamin C, 2.5% N- acetyl-L-glutamines, 1.5% branched-chain amino acid leucine, surplus are trained for cell
Support base, cell culture medium is existing culture medium on the market, preferably gibic, hyclone, lonza, stemcell, BI,
DMEM culture mediums, H3 serum free mediums or the RPMI-1640 culture mediums of sigma or takara companies.
Embodiment 2
The purpose of the present embodiment is that providing a kind of cell culture fluid, mainly it is grouped as by the group of following volumes percentage：
2.5% vitamin C, 2.5% N- acetyl-L-Histidine, 1.5% branched chain amino acids valine, surplus are cell culture
Base, cell culture medium are existing culture medium, preferably gibic, hyclone, lonza, stemcell, BI, sigma on the market
Or DMEM culture mediums, H3 serum free mediums or the RPMI-1640 culture mediums of takara companies.
Embodiment 3
The purpose of the present embodiment is that providing a kind of cell culture fluid, mainly it is grouped as by the group of following volumes percentage：
2.5% vitamin C, 2.5% N- acetyl-L-lysine, 1.5% branched-chain amino acid isoleucine, surplus are trained for cell
Support base, cell culture medium is existing culture medium on the market, preferably gibic, hyclone, lonza, stemcell, BI,
DMEM culture mediums, H3 serum free mediums or the RPMI-1640 culture mediums of sigma or takara companies.
Three kinds of common amino acids in protein, leucine, valine and isoleucine are referred to as branched-chain amino acid
(BCAA), so compound branched-chain amino acid can be claimed again, wherein optimal with leucine.This amino acid is promoted with two kinds of particular forms
Anabolism (muscle growth)：1. promoting insulin releasing, 2. promote growth hormone release.In branched-chain amino acid most importantly
Leucine.In addition to above-mentioned three kinds of N- acetylamino acids, l-Isoleucine, L-Leu, L-Methionine, L- phenylpropyl alcohols can also be used
Propylhomoserin, L-threonine, L-Trp or L- valines are the bioactive substances for maintaining cell growth, are risen in cell metabolism
Adjusting and control action.
The cell growth state of cell culture fluid culture provided by the present invention is good, can be used on the market a variety of at present
The culture of cell line and some more difficult primary cells captured.
Embodiment 4
The purpose of the present embodiment is that provide a kind of platelet rich plasma, the platelet rich plasma prepared by following steps and
At (following culture operation, which is placed on aseptic operating platform, to be operated)：
S1：The peripheral blood 100ml of human body is taken, respectively to 10 15ml cell culture tubes (eagle medium-thermo
Factory produces) in, in advance added with anti-coagulants in cell culture tube, it is added described in 0.5mL embodiments 1 in each cell culture tube
Culture solution, mixing obtain mixed liquor one；
S2：By mixed liquor one, stationary culture, the temperature of culture are 37 DEG C in cell incubator, CO when culture2It is a concentration of
5%, incubation time 4h obtain mixed liquor two；
S3：Mixed liquor two described in step S2 is centrifuged, centrifugal force 26G, centrifugation time 15min, take supernatant and
Intermediate cell layer is to get to the platelet rich plasma.
In the platelet rich plasma preparation process, the intermediate cell layer obtained by step S3 includes a large amount of leucocyte, blood
Platelet and a certain amount of mescenchymal stem cell, all acellular components in supernatant in the blood plasma containing self whole blood, example
Such as cell factor, growth factor, chemotactic factor (CF), immune-regulating factor, enzyme, hormone；Growth factor includes insulin-like growth
Factor IGF-1, transforming growth factor TGF-β, alkalinity are in fibroblast growth factor FGF-b and vascular endothelial growth factor
VEGF etc..Due to platelet rich plasma preparation method provided by the invention, first carried out using cell culture fluid provided by the invention
Culture, makes the cell in peripheral blood as often as possible discharge beneficial acellular components, makes these beneficial acellular components, mainly
It is all kinds of growth factor fast activating hyperplasia, after the completion of culture, is once centrifuged, the platelet rich plasma collected contains
The quantity of all kinds of growth factors be significantly larger than the active ingredient in the platelet rich plasma that conventional method prepares, and should
For cultural method merely through primary centrifugation, intermediate steps are few, and whole process operates on aseptic operating platform, safety higher, richness obtained
Thrombocyte plasma can be directly injected into patient body.It can be extracted out per 100mL peripheral bloods total about comprising supernatant and intermediate cell layer
The liquid of 40mL, is injected into scalp and can be used to hair tonic.Centrifuge used is kubota2800 type centrifuges.
Effect example 1
100 alopecia seborrheica patients (62 patients of universality alopecia, seriousness hair loss patient 38) are collected, are extracted
The peripheral blood of aforementioned patient collects the rich blood of each patient according to the preparation method culture for implementing the platelet rich plasma described in 3
Platelet-poor plasma, and the platelet rich plasma of collection is corresponded to and is injected in the scalp of each patient, universality hair loss patient and serious
Property hair loss patient start hair tonic in three months after 1 treatment, hair tonic rate reaches 100%, i.e., three after 1 time treatment
Hair can be grown in month.Treatment cycle varies with each individual, and is learnt by practice, according to platelet rich plasma provided by the invention
Platelet rich plasma made from preparation method, new hair tonic coverage rate (new life fermentation product and alopecia area ratio) as shown in table 1,
It is that can reach 90% or more (universality hair loss patient that universality hair loss patient, which generally passes through the 3-4 new hair tonic coverage rate for the treatment of,
Therapeutic effect figure as shown in Fig. 2, the figure that wherein number is 1-4 indicates treatment respectively before, third is all after seance, Retreatment
Third week and the three times design sketch in treatment third week afterwards), the new hair tonic covering generally after 6-9 treatment of seriousness hair loss patient
Rate can reach 90% or more, and (the therapeutic effect figure of seriousness hair loss patient is as shown in Figure 1, the figure difference that wherein number is 1-4
Indicate treatment before, after seance, after Retreatment and six times treatment design sketch).
Table 1
It can be seen that the cell and growth factor in the platelet rich plasma that embodiment 2 provides can stimulate seborrheica take off
The hair follicle for sending out patient's suspend mode aging, allows the hair tonic again of these hair follicles, and hair tonic rate is 100% after treating for the first time, and the present invention carries
The cultural method of the platelet rich plasma of confession is different from general hair growth treatment or hair tonic medicament, and traditional hair growth treatment is mostly to plant
Hair, can not effect a radical cure alopecia, platelet rich plasma provided by the invention can fundamentally treat patient's alopecia, it is made constantly to grow newly
Hair.
The present invention is not limited to above-mentioned optional embodiment, anyone can show that other are each under the inspiration of the present invention
The product of kind form.Above-mentioned specific implementation mode should not be understood the limitation of pairs of protection scope of the present invention, protection of the invention
Range should be subject to be defined in claims, and specification can be used for interpreting the claims.
Claims (8)
1. a kind of cell culture fluid, which is characterized in that be mainly grouped as by the group of following volumes percentage：2.5% vitamin
C, 2.5% N- acetylamino acids, 1.5% branched-chain amino acid, surplus are cell culture medium.
2. cell culture fluid according to claim 1, it is characterised in that：Amino acid in the N- acetylamino acids is L-
Methionine, L-threonine, L-Histidine, L-Glutamine, l-Isoleucine, L-Leu, L-lysine, L-phenylalanine,
L-Trp or Valine, the branched-chain amino acid are leucine.
3. cell culture fluid according to claim 1 or 2, it is characterised in that：The cell culture medium be DMEM culture mediums,
H3 serum free mediums or RPMI-1640 culture mediums.
4. a kind of preparation method of platelet rich plasma, which is characterized in that include the following steps：
S1：Peripheral blood is taken, the cell culture fluid described in claim 1-3 any one is added into peripheral blood, mixing is mixed
Close liquid one；
S2：By one stationary culture of above-mentioned mixed liquor, mixed liquor two is obtained；
S3：Mixed liquor two described in step S2 is centrifuged, takes supernatant and intermediate cell layer to get to the rich platelet blood
Slurry.
5. preparation method according to claim 4, it is characterised in that：In the step S1 volume of peripheral blood with it is described thin
The ratio of born of the same parents' culture solution is 20:1.
6. preparation method according to claim 5, it is characterised in that：The temperature cultivated in the step S2 is 37 DEG C, training
CO when supporting2A concentration of 5%, incubation time 4h.
7. preparation method according to claim 6, it is characterised in that：Centrifugal force is 26G when being centrifuged in the step S3, from
The heart time is 15min.
8. platelet rich plasma prepared by a kind of preparation method by described in claim 4-7 any one.
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Cited By (1)
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| CN110638833A (en) * | 2019-11-15 | 2020-01-03 | 西安圣德生物科技有限公司 | Composition for promoting hair growth and method of use thereof |
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Application publication date: 20181113 |