NL2032529B1

NL2032529B1 - Method for preparing platelet growth factor concentrate by kit

Info

Publication number: NL2032529B1
Application number: NL2032529A
Authority: NL
Inventors: Shan Letian; Zhou Li
Original assignee: Univ Zhejiang Chinese Medical
Priority date: 2022-07-18
Filing date: 2022-07-18
Publication date: 2024-01-26

Abstract

The present invention provides a method for preparing a platelet growth factor concentrate (GF—PL) by a kit, and belongs to the technical field of regenerative medicine. According to the method, anticoagulant whole blood is kept static for 30—60 min, and then 5 centrifuged by a platelet—rich plasma preparation kit, so that the purity and yield of products can be improved; after centrifugation, a platelet—rich plasma (PRP) layer and a platelet— poor plasma (PPP) layer are retained and freeze—thawed, so that growth factors can be obtained maximally. The GF—PL prepared by 10 the method contains high concentrations of various growth factors, including PDGF, TGF—ß, IGF—l, EGF, FGF and VEGF, and the concentration of each factor is significantly higher than that in a product (PRP) prepared, by a conventional platelet kit and a product prepared by conventional platelet lysate, and the results 15 of differences are statistically significant. (+ Fig. l)

Description

P1479/NLpd

METHOD FOR PREPARING PLATELET GROWTH FACTOR CONCENTRATE BY KIT

TECHNICAL FIELD

The present invention relates to the technical field of re- generative medicine, in particular to a method for preparing a platelet growth factor concentrate by a kit.

BACKGROUND ART

Platelet-rich plasma (PRP) is a platelet concentrate obtained from autologous anticoagulant whole blood after centrifugation.

PRP promotes tissue regeneration and repair, including articular cartilage repair, by releasing various growth factors (such as

PDGF, IGF-1, EGF, FGF, TGF-beta and VEGF). The higher content of growth factors in PRP, the more conducive to regenerating and re- pairing tissue. Therefore, a platelet concentrate containing a high concentration of growth factors is needed at present.

SUMMARY

The present invention is intended to provide a method for preparing a platelet growth factor concentrate (GF-PL) by a kit, and the GF-PL has high growth factor contents, which are conducive to regenerating and repairing tissue.

To achieve the aforesaid purpose, the present invention pro- vides the following technical solutions:

The present invention provides a method for preparing a GF-PL by a kit, including the following steps: 1) keeping anticoagulant whole blood static for 30-60 min, centrifuging the anticoagulant whole blood by a platelet-rich plasma preparation kit, and collecting products of a platelet-rich plasma (PRP) layer and a platelet-poor plasma (PPP) layer to ob- tain centrifuged products; 2) freeze-thawing the centrifuged products 3-6 times to ob- tain frozen-thawed products; 3) centrifuging the frozen-thawed products, and collecting supernatant to obtain a GF-PL.

Preferably, a number of times of the freeze-thawing in 32) is 4-5.

Preferably, a method of the freeze-thawing in 82) includes liquid nitrogen freeze-thawing.

Preferably, the centrifuged products are frozen at -20--196°C for 5-10 min and then thawed at 4-37°C for 5-10 min in each freeze- thawing process in S2).

Preferably, the range of the freezing temperature in S2) is - 30--80°C.

Preferably, a process of the thawing is accompanied by ultra- sonic or oscillation treatment.

Preferably, the range of the centrifugation speed in S3) is 3,000-10,000 rpm, the range of the centrifugation time is 5-10 min, and the range of the centrifugation temperature is 0-4°C.

The present invention further provides a GF-PL prepared by the preparation method in the above-mentioned solutions.

The present invention further provides the application of the

GF-PL in the above-mentioned solutions in preparation of tissue regeneration and repair drugs.

Preferably, the tissue regeneration and repair include artic- ular cartilage repair, skin wound repair and pressure ulcer injury repair.

Beneficial effects of the present invention: The present in- vention provides a method for preparing a GF-PL by a kit. Accord- ing to the method, anticoagulant whole blood is kept static for 30-60 min, and then centrifuged by a PRP preparation kit, so that the purity and yield of products can be improved; after centrifu- gation, a PRP layer (a middle layer) and a PPP layer (an upper layer) are retained and freeze-thawed, so that growth factors can be obtained maximally. The GF-PL prepared by the method contains high concentrations of various growth factors, including PDGF,

TGF-B, IGF-1, EGF, FGF and VEGF, and the concentration of each factor is significantly higher than that in a product (PRP) pre- pared by a conventional platelet kit and a product prepared by conventional platelet lysate (PL), and the results of differences are statistically significant.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows concentrations of various growth factors in GF-

PL and PRP as tested by ELISA in example 2 and comparisons, where

A represents a content of PDGF, B represents a content of TGF-$, C represents a content of IGF-1, D represents content of EGF, E rep- resents a content of FGF, and F represents a content of VEGE;

P<0.01 indicates that concentrations between GF-PL and PRP are significantly different.

FIG. 2 shows general observation results of nude mouse models in example 3, where A represents a normal group, B represents a model group, C represents a GF-PL intervention group, D represents a PRP intervention group, E is a microscopic magnification image of neck and back skins of the normal group, F is a microscopic magnification image of neck and back skins of the model group, G is a microscopic magnification image of neck and back skins of the

GF-PL group, and H is a microscopic magnification image of neck and back skins of the PRP intervention group.

FIG. 3 shows histopathological observation results of skins of nude mice by Masson staining in example 3, where A represents a

PRP intervention group, B represents a GF-PL intervention group, C represents a model group, and D represents a normal group.

FIG. 4 shows dermal thickness measurement results of skins of nude mice in example 3.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The present invention provides a method for preparing a GF-PL by a kit, including the following steps: 1) keeping anticoagulant whole blood static for 30-60 min, centrifuging the anticoagulant whole blood by a PRP preparation kit, and collecting products of a PRP layer (a middle layer) and a

PPP layer (an upper layer) to obtain centrifuged products; 2) freeze-thawing the centrifuged products 3-6 times to ob- tain frozen-thawed products; 3) centrifuging the frozen-thawed products, and collecting supernatant to obtain a GF-PL.

According to the method, anticoagulant whole blood is kept static for 30-60 min, the anticoagulant whole blood is centrifuged by a PRP preparation kit, and products of a PRP layer (a middle layer) and a PPP layer (an upper layer) are collected to obtain centrifuged products. In the specific implementation process, the anticoagulant whole blood is collected by a conventional method; the range of the static keeping temperature is preferably 18-25°C; the PRP preparation kit is common commercially available; in the specific implementation process, a PRP preparation kit (GXZZ 20163661321) (CN 105107233 A) was selected, which was purchased from Shandong Wego New Life Medical Devices Co., Ltd.

After the centrifuged products are obtained, they are frozen- thawed 3-6 times to obtain frozen-thawed products. A number of times of the freeze-thawing is preferably 4-5; and a method of the freeze-thawing preferably includes liquid nitrogen freeze-thawing.

The centrifuged products are frozen at -20--196°C for 5-10 min and then thawed at 4-37°C for 5-10 min in each freeze-thawing process; and the range of the freezing temperature is preferably -30--80°C.

A process of the thawing is preferably accompanied by ultrasonic or oscillation treatment; the ultrasonic or oscillation treatment is beneficial to release growth factors and improve the freeze- thawing efficiency and the extraction rate of growth factors. The ultrasonic treatment is preferably performed in an ultrasonic thermostat water bath; the ultrasonic power is preferably 500-600

W; and the ultrasonic frequency is preferably 40-50 KHZ. The os- cillation treatment is preferably performed in a constant tempera- ture oscillating metal bath; the oscillation speed is preferably 1,800 rpm; the oscillation power is preferably 200 W; and the os- cillation amplitude is a 3 mm horizontal rotation.

The conventional PRP plays its role by gradually releasing growth factors from platelets. However, in the present invention, upon the freeze-thawing treatment for 3-6 times, a GF-PL is ob- tained from PRP+PPP lysate, so that these growth factors can be directly obtained, and concentrations are higher than those in the conventional PRP.

After that, the frozen-thawed products are centrifuged, and supernatant is collected to obtain a GF-PL; wherein a speed of the centrifugation is preferably 3,000-10,000 rpm, a number of is preferably 5-10 min, and a temperature is preferably 0-4°C; the function of the centrifugation is to remove cell debris and other impurities. 5 The present invention further provides a GF-PL prepared by the method in the above-mentioned solutions.

The present invention further provides the application of the

GF-PL in the above-mentioned solutions in preparation of tissue regeneration and repair drugs; the tissue regeneration and repair preferably include articular cartilage repair, skin wound repair and pressure ulcer injury repair.

Example 1 1) Anticoagulant whole blood collected was kept static for 30 min, the anticoagulant whole blood was centrifuged by a PRP prepa- ration kit (GXZZ 20163661321; purchased from Shandong Wego New

Life Medical Devices Co., Ltd.; CN 105107233 A), and products of a

PRP layer and a PPP layer (products of PRP+PPP) were collected to obtain centrifuged products; 2) the centrifuged products were frozen-thawed 5 times be- tween 37--80°C (a freezing temperature was -20--80°C, a thawing temperature was 4-37°C, and a thawing process was performed in an ultrasonic water bath) to obtain frozen-thawed products; 3) the frozen-thawed products were centrifuged (5,000 rpm, 10 min, 4°C), and supernatant was collected to obtain a GF-PL.

Comparative example 1

Anticoagulant whole blood collected was centrifuged by a PRP preparation kit (GXZZ 20163661321; purchased from Shandong Wego

New Life Medical Devices Co., Ltd.; CN 105107233 A), and a PRP layer was collected to obtain PRP.

Example 2 Comparisons between the GF-PL prepared in example 1, the PRP prepared by the preparation method of the kit in com- parative example 1 and conventional PL (by reference to Patent 104673747B)

Method: All samples to be tested were prepared, wells of an

ELISA plate were immersed in 300 pl of washing solution, standard substances and samples (100 pl) were added to different wells re-

spectively after the wells were spin-dried, then 50 pl of testing antibody was added to each well, and the plate was sealed with a microplate sealer to oscillate at 300 rpm and incubate at a room temperature for 2 h; the plate was washed with washing solution and then enzyme (horseradish peroxidase marked streptavidin) was added, and the plate was sealed with a new microplate sealer to oscillate at 300 rpm and incubate at a room temperature for 45 min; the plate was washed with washing solution and then 100 pl of chromogenic substrate TMB was added to each well, and the plate was incubated away from light at a room temperature for 5-30 min; finally 100 pl of stop solution was added to each well, observing the samples changed from blue to yellow, then subjected to dual- wavelength detection by a microplate reader, and OD values at 450 nm maximum absorption wavelength and 570 nm or 630 nm reference wavelength were determined. Result analysis: Average OD values of the standard substances and the samples were calculated, then OD values of zero concentration standard substances were subtracted, regression fitting was performed to generate standard curves, and a concentration of each growth factor was obtained by logarithmic fitting of concentration values and OD values. Each sample was de- tected 3 times.

Results: The ELISA results show that (in FIG. 1, A represents

PDGF, B represents TGF-$8, C represents IGF-1, D represents EGF, E represents FGF, and F represents VEGF), the GF-PL obtained by the method of the present invention contains high concentrations of various growth factors, including PDGF, TGF-8, IGF-1, EGF, FGF and

VEGF, and the concentration of each factor is significantly higher than that in the product (PRP) prepared by a conventional platelet kit and in the PL group (**P<0.01 represents a comparison between groups, indicating that the contents of growth factors in the PRP and PL groups are extremely significantly lower than those in the

GF-PL group of the present invention), and the results of differ- ences are statistically significant.

Example 3 Comparisons between application of the GF-PL pre- pared in example 1 and application of the PRP prepared by the preparation method of the kit in comparative example 1 in skin wound repair

Modeling and intervention: 40 6-week-old SPF male nude mice were taken and randomly di- vided into 5 groups: normal group, model group, low-concentration

GF-PL group, GF-PL group and PRP group, 10 mice in each group. Ex- cept for the normal group, the animals in other groups were in- jected subcutaneously with D-gal (1,000 mg/kg, subcutaneously) on the backs every day for 8 weeks. The left and right sides of the backs could be compared. All animals were allowed to access water and food freely, and changes in the skins at the injection sites on the backs were observed daily. Photos were taken for recording.

From the 8" week, the GF-PL group and the PRP group were injected with GF-PL (107/ml) and PRP (107/ml) respectively at the same site every other week, for totally 4 weeks. The control group was in- jected with normal saline. At the 12% week, the mice were killed, and small pieces of skins in the same areas on the backs were tak- en for histological examination and Masson staining.

Results:

The general observation results are shown in FIG. 2, where A represents a normal group, B represents a model group, C repre- sents a GF-PL intervention group, D represents a PRP intervention group, E is a microscopic magnification image of back skins of the normal group, F is a microscopic magnification image of back skins of the model group, G is a microscopic magnification image of back skins of the GF-PL intervention group, and H is a microscopic mag- nification image of back skins of the PRP intervention group. Af- ter modeling, the model group showed obvious skin wrinkles, but after the intervention of GF-PL and PRP, the wrinkles were signif- icantly relieved. The wrinkles of the nude mice in the GF-PL group were less than those in the PRP group. The histopathological ob- servation is shown in FIG. 3, where A represents a PRP interven- tion group, B represents a GF-PL intervention group, C represents a model group, and D represents a normal group. The dermal thick- ness measurement results are shown in FIG. 4. In the normal group, the epidermis is intact, the dermis is complete, and the staining is normal. In the model group, the dermis becomes thinner and the staining is shallow, indicating that the dermal tissue and colla- gen content decreased. In both GF-PL and PRP groups, the dermis and collagen content are higher than those in model group, and the dermal thickness in GF-PL group is significantly higher than that in PRP group.

These results show that both GF-PL and PRP have the function of aged skin repair, and the GF-PL is obviously superior to the PRP.

Claims

CONCLUSIONS

1. Method for preparing a platelet growth factor concentrate (GF-PL) by means of a kit, comprising the following steps: 1) keeping anticoagulated whole blood static for 30 to 60 minutes, centrifuging the anticoagulated whole blood with a platelet-rich plasma preparation kit, and collecting products from a platelet-rich plasma (PRP) layer and a platelet-poor plasma (PPP) layer to obtain centrifuged products; 2) freezing and thawing the centrifuged products 3 to 6 times to obtain frozen-thawed products; 3) centrifuging the frozen-thawed products, and collecting supernatant to obtain a GF-PL.

The preparation method according to claim 1, wherein a number of times of the freeze-thaw treatment in S2) is 4 to 5.

Preparation method according to claim 1, wherein a method for the freeze-thaw treatment in S2) comprises freeze-thaw with liquid nitrogen.

The preparation method according to claim 1, wherein the centrifuged products are frozen for 5 to 10 minutes at -20 to -196°C and then thawed for 5 to 10 minutes at 4 to 37°C in each freeze-thaw treatment in S2).

5. Preparation method according to claim 4, wherein the freezing temperature range is -30 to -80°C.

Preparation method according to claim 4, wherein a thawing method involves ultrasonic or oscillating treatment.

The preparation method according to claim 1, wherein the range of centrifugation speed in 83) is 3000 to 10,000 rpm, the range of centrifugation time is 5 to 10 min, and the range of centrifugation temperature is 0 to 4°C.

8. GF-PL prepared according to the preparation method according to any one of claims 1 to 7.

Use of the GF-PL according to claim 8 in the preparation of tissue regeneration and repair drugs.

Use according to claim 9, wherein the tissue regeneration and repair include the repair of articular cartilage, the repair of skin wounds and the repair of pressure ulcers.