CN110846274A

CN110846274A - Preparation method of compound cell preparation applied to osteoarthritis and use method of compound cell preparation

Info

Publication number: CN110846274A
Application number: CN201911143931.6A
Authority: CN
Inventors: 谢海涛; 王斌; 薛卫巍; 方晓; 毛文哲
Original assignee: Guangdong Xiankangda Biotechnology Co Ltd
Current assignee: Guangdong Xiankangda Biotechnology Co Ltd
Priority date: 2019-11-20
Filing date: 2019-11-20
Publication date: 2020-02-28

Abstract

The invention discloses a preparation method of a compound cell preparation applied to osteoarthritis, which comprises the following steps: and mixing the activated adipose-derived stem cells with platelet-rich plasma to prepare a composite cell preparation, wherein the concentration of the activated adipose-derived stem cells is 4-6 million per milliliter, and the concentration of platelets is 3-5 million per milliliter. The invention also discloses an application method of the compound cell preparation in osteoarthritis, which comprises the following steps: firstly, injecting a certain amount of activated adipose-derived stem cells into a joint cavity, and then injecting a compound cell preparation into the joint cavity, wherein the dosage is controlled to be 1.5-3 ml/time, 1-2 times per month, and the injection lasts for 2-6 months. The invention has the advantages of low cost, short treatment course and good curative effect.

Description

Preparation method of compound cell preparation applied to osteoarthritis and use method of compound cell preparation

Technical Field

The invention relates to the field of medicines, in particular to a preparation method of a compound cell preparation which is low in cost, short in treatment course and good in curative effect and is applied to osteoarthritis, and a use method of the compound cell preparation.

Background

The cartilage regeneration technology is an orthopedic problem, and the reason is that the cartilage regeneration technology is used for the following aspects: (1) articular cartilage has no self-repairing ability, (2) chondrocytes cannot migrate to the defect site, and (3) tissues have no blood supply and no innervation. Although the regeneration of cartilage is difficult, the repair is still attempted, and in modern medicine, the cartilage repair technology is still silently developed as a medical technology which can benefit a large number of patients. According to the chronological order of development, the method can be divided into the following four generations:

first generation: bone marrow stimulation techniques

Because the articular cartilage has no blood vessels and nerves distributed therein and lacks self-repairing capability, the orthopedics doctor needs to make several holes at the diseased part to allow the bone marrow to ooze. The fibrocartilage can be formed by covering the surface of the damaged cartilage with blood from bone marrow and stem cells having a strong ability to regenerate in the blood. The technology is simple and effective, and has good short-term effect. However, the newly formed repair tissue is not a real articular cartilage, and this tissue hardens into an osteogenic tissue in about 5 years, losing the function of cartilage, and further accelerating the progression of the disease.

And (4) second generation: cartilage transplantation technique

This technique is divided into autologous transplantation and embedded transplantation. Autografting refers to a technique of obtaining a cartilage column from a non-load bearing area of an ipsilateral joint and implanting the cartilage column into a square hole at a cartilage defect. However, the number and size of the obtained autologous chondrocytes are limited. The technique of embedded grafting is a technique of embedding a plurality of smaller cartilage columns into a cartilage defect. However, due to the insertion of the plurality of tapered cartilage columns, the cartilage surface facing the joint cavity becomes uneven, thus initiating a new cycle of wear osteoarthritis pathology.

And a third generation: cartilage cell planting technology

The currently most widely used method of cartilage regeneration is autologous chondrocyte seeding technology. The technology firstly obtains chondrocytes from a non-load bearing area of an ipsilateral joint, then cultures in vitro, and finally plants the chondrocytes at a cartilage defect to generate hyaline cartilage which is closer to the structural and functional characteristics of normal cartilage.

It solves the problem of 'quantity shortage' of the second generation technology and the difficult point of 'not long time' of the first generation technology. However, the technology is also under continuous development and improvement, and the application of the technology to the femoral side cartilage defect achieves more ideal effect at present, and is particularly suitable for young patients with meniscus tear and ligament instability. The technique needs 2 operations, and has long recovery period, which brings pain and inconvenience to patients.

Fourth generation: stem cell therapy techniques

In the field of relatively hot research at the present stage, the fat stem cells or umbilical cord mesenchymal stem cells are used, and the osteoarthritis treatment effect is remarkable. In recent years, relevant basic researches of stem cell source conditioned medium, extracellular matrix, exosome and microvesicle in the field of knee osteoarthritis and cartilage repair are consulted and summarized. As a result, it has been found that the therapeutic effects of stem cells and paracrine effects on knee osteoarthritis and articular cartilage damage have been revealed in various studies. The action mechanism of the medicine comprises effective inhibition on inflammatory reaction in joint cavity, chondrocyte apoptosis and cartilage matrix hydrolysis, and repair processes of promoting cartilage matrix synthesis, directional differentiation of in-situ inherent stem cells to chondrocytes, directional migration to damaged parts and the like. The curative effect can be further improved by combining the means of tissue engineering method or gene modification and the like.

In summary, the cartilage regeneration technology and the development of the current generation 4 are one-pass in each generation, but problems of each generation are discovered with the lapse of time. The newly formed repair tissue of the 1 st generation technology is not real articular surface cartilage, and the tissue can harden osteogenic tissue in about 5 years, lose the function of the cartilage and further accelerate the progress of diseases. The 2 nd generation technology, due to the insertion of a plurality of tapered cartilage columns, the cartilage surface facing the joint cavity becomes uneven, thus initiating a new cycle of wear osteoarthritis pathology. The 3 rd generation technique requires 2 operations and has a long recovery period, which brings pain and inconvenience to patients. The 4 th generation technology stem cells can not be expressed for a long time within the effective survival time in the joint cavity. The survival time of stem cells after intravenous injection is about 10-48 hours according to the fluorescent labeling of cells, and 80% of the survival time is mainly concentrated in the lung and the pancreas. The survival rate of stem cells can be increased by more than 60% through local injection, and the survival time is about 15 days. In a short time, stem cells need to be activated and induced to differentiate into protochondrocytes, which are less efficient and cannot be expressed for a long time.

Disclosure of Invention

In view of the above, the present invention aims to provide a method for preparing a compound cell preparation with low cost, short treatment course and good curative effect for osteoarthritis, which aims at solving the problems of the existing cartilage regeneration technology.

The purpose of the invention is realized by adopting the following technical scheme:

a preparation method of a compound cell preparation applied to osteoarthritis comprises the following steps:

and mixing the activated adipose-derived stem cells with platelet-rich plasma to prepare a composite cell preparation, wherein the concentration of the activated adipose-derived stem cells is 4-6 million per milliliter, and the concentration of platelets is 3-5 million per milliliter.

Optionally, the preparation method of the activated adipose-derived stem cells comprises:

preparing a DMEM medium, wherein the proportion of the DMEM medium is 1% fetal calf serum, 10ng/ml TGF- β, 100ng/ml BMP-6, 6.25 mu g/ml insulin, 0.1 mu mol/L dexamethasone, 6.25 mu g/ml transferrin and 50 mu mol/L ascorbic acid phosphate;

placing the adipose-derived stem cells forming the micelles in a DMEM culture medium for culturing;

injecting the adipose-derived stem cells cultured for 7-9 days into joint cavity to transform into protochondrocytes.

Optionally, the preparation steps of the adipose-derived stem cells are as follows:

cleaning adipose tissues by using a buffer solution to remove residual blood and tissue fragments, cutting the cleaned adipose tissues into small pieces, digesting the small pieces in a shaking box for 20-60 min, standing the small pieces until the small pieces are layered, sucking upper layer adipose cell fluid, placing the adipose cell fluid in a box body at the temperature of 0-5 ℃ for culturing for 1-2 hours, sealing and centrifugally separating the box body, and removing supernatant to prepare adipose stem cell suspension;

placing the adipose-derived stem cell suspension in a cell activation medium for activation culture for 10-20 hours so as to obtain preliminarily expanded adipose-derived stem cells;

placing the adipose-derived stem cells into at least one cell culture medium and adding an activating reagent to perform adipose-derived stem cell suspension culture for 12-15 hours so as to obtain activated adipose-derived stem cells;

taking out the activated adipose-derived stem cells from the culture medium, adding normal saline, cleaning, adding enzyme solution for digestion, and culturing for three to six generations after the adipose-derived stem cells grow to 80-90% and are fused;

and naturally placing the culture solution after passage for 3-5 hours to obtain the micelle adipose-derived stem cells.

Optionally, the adipose-derived stem cells are cultured for five generations after 80% -90% of the adipose-derived stem cells are fused.

Optionally, the cell activation medium is a serum-free lymphocyte culture medium added with interleukin-2 and saperin.

Optionally, the preparation method of the platelet-rich plasma comprises the following steps:

collecting blood samples and adding anticoagulant, performing primary centrifugation and collecting liquid without blood cells on the upper layer, performing secondary centrifugation on the liquid by using centrifugal force 4-7 times of the primary centrifugal force, and sucking upper layer plasma until the remaining bottom liquid is 4-5% of the volume of the blood samples to prepare platelet-rich plasma.

Optionally, the anticoagulant is citric acid, the preparation volume concentration is 0.109 mol/L-0.129 mol/L, and the volume ratio of the anticoagulant to the blood sample is 1: 9.

optionally, the preliminary centrifugation is 200g and 8min, and the secondary centrifugation is 1000g and 10 min.

Optionally, the anticoagulant is prepared by hydrogen-rich water.

The invention also discloses an application method of the compound cell preparation in osteoarthritis, which comprises the following steps:

firstly, injecting a certain amount of activated adipose-derived stem cells into a joint cavity, and then injecting the compound cell preparation prepared according to any one of claims 1 to 9 into the joint cavity, wherein the dosage is controlled to be 1.5-3 ml/time, 1-2 times per month, and 2-6 months.

In order to fully utilize the limited time and activate the ASCs to transform to the chondrocytes as soon as possible, the invention creatively invents that the ASCs are activated only in vitro while ensuring the original activity of the ASCs, the ASCs are stimulated to an activated state, the process of converting to a chondrocyte direction is ensured, the time point and the strength of activation are adjusted, and the ASCs in the activated state are injected into a diseased joint cavity to continue to transform to the chondrocytes according to the original plan. This saves time and no longer emphasizes the use of three-dimensional models and a more difficult to control augmented growth environment, and the resulting chondrocytes also reduce the corresponding rejection response.

Detailed Description

It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

The method comprises the steps of firstly preparing activated adipose stem cells, obtaining adipose tissues to be treated according to the method from a patient or an immunologically acceptable donor, separating the adipose tissues from an adipose sample, culturing the activated adipose tissues, wherein the "immunologically acceptable donor" is a human with tissues and comprises adipose tissues, firstly washing the adipose tissues by using a buffer solution to remove residual blood and tissue fragments, washing the washed adipose tissues by using a D-Hanks buffer solution in the embodiment, shearing the washed adipose tissues into small pieces, digesting the small pieces in a shaking box for 20 to 60min, standing the small pieces until the layering, sucking upper layer adipose cell fluid, placing the adipose cell fluid in a box at 0 to 5 ℃ for 1 to 2 hours, finding that the activity of the adipose cell fluid can be increased by using the previous box at 0 to 5 ℃ for 1 to 2 hours, sealing and centrifuging the separated cells, removing supernatant, preparing adipose stem cells, placing the adipose cell fluid in a cell activation culture medium to perform activation culture for 10 to 20 hours so as to obtain the expanded adipose stem cells, and placing the adipose stem cells in a cell activation medium for 10 to perform activation culture by using a serum-supplemented medium, preferably a serum-supplemented bovine serum-supplemented animal serum-supplemented medium, preferably a serum-supplemented medium for 2% serum-supplemented bovine serum-supplemented animal serum-supplemented medium for at least 10 serum-supplemented animal serum-supplemented medium for 2 hours, preferably for 2% serum-supplemented animal stem cells, preferably for 2 hours, preferably for 2-bovine serum-supplemented animal stem cells, preferably for 2 hours, and bovine serum-supplemented animal stem cells, preferably for 2 hours, or bovine serum-supplemented animal stem cells, preferably for 2 serum-bovine serum-supplemented animal stem cells, preferably for 2-bovine serum-supplemented animal stem cells, and bovine serum-supplemented for 2 hours, or bovine serum-supplemented animal stem cells, preferably for 2 hours, and bovine serum-supplemented for 2-bovine serum-supplemented for at least one or bovine serum-supplemented for 2 hours, preferably for at least one or bovine serum-supplemented for at least one or bovine serum-supplemented for 2 hours, preferably for at least one or bovine serum-supplemented animal stem cells, for about 10 serum-supplemented animal stem cells, for about 10 serum-supplemented for about 10 hours, preferably for about 10 hours, for about 10 serum-supplemented for about 10 hours, preferably for about 10 hours, for about 10 days.

The inventor creatively invents that the ASCs are only activated in vitro while the original activity is ensured, the ASCs are stimulated to be in an activated state, the process of converting towards a cartilage cell direction is ensured, the time point and the intensity of activation are adjusted, and the ASCs in the activated state are injected into a diseased joint cavity to be continuously converted into the cartilage cell according to the original plan. This saves time without emphasizing the use of a three-dimensional model and a more difficult to control augmented growth environment, and the resulting chondrocytes also reduce the corresponding rejection response.

In the preparation of the adipose-containing stem cells related to the invention, the inventor finds that after calcium ionophore or adenovirus is added into the adipose-containing stem cells, the activated adipose-containing stem cells can stimulate the secretion amount of exosomes after entering a human body, which is 2-3 times of the secretion amount of other culture modes. The adipose-derived stem cells can be activated by contacting them with an activating agent, and the activated adipose-derived stem cells have a functional level that stimulates secretion, and the secretion amount of the exosomes is further increased by further subculturing.

Calcium ionophore is a specific metal ion species that can freely pass through lipid bilayers and soluble lipids. There are two types of ionophores: ions formed by the carrier or the channel, like adenovirus is used as the carrier, a cage-shaped structure is formed around the special ions, and the ions can freely diffuse in a hydrophobic region of a hydrophobic bilayer; channel-forming ions, such as gram-positive bacteria, form continuous liquid polar surfaces in a bilayer molecular membrane, allowing ions to diffuse through. In addition, suitable ionophores for the present invention include calcium ionophore A23187 (calcimycin), sodium salts, magnesium salts, and the like, in addition to the carriers described above. Such as calcium ionophore a23187, which are capable of responding to changes in PH gradients to concentrate calcium ions. Calcium ionophore a23187 has an acidic carboxyl group which exchanges with other cations throughout the biofilm and returns to the other end of the membrane when ion exchange is complete. The effective concentration of the ionophore is 0.05-0.5 ug/ml, and the effective concentration of the ionophore is the effective concentration for activating the adipose-derived stem cells.

Preparing plasma rich in platelets, collecting a blood sample, and adding an anticoagulant, wherein the anticoagulant can be selected from ethylenediamine tetraacetic acid or salts thereof, citric acid glucose solution, and ethylenediamine tetraacetic acid or salts thereof, the anticoagulant in the embodiment is citric acid, the volume concentration of the prepared anticoagulant is 0.109 mol/L-0.129 mol/L, and the volume ratio of the anticoagulant to the blood sample is 1: 9; primarily centrifuging and collecting the upper layer of liquid without blood cells, and centrifuging the liquid again by a centrifugal force 4-7 times of the primary centrifugal force, wherein in the embodiment, about 40-50 ml of a blood sample (with an anticoagulant) is collected and centrifuged for 200g and 8 min; layering after centrifugation is finished, collecting and counting the upper layer of plasma, wherein the upper layer of plasma is plasma and the lower layer of red blood cells, about 80% of platelets can be collected, and blood cells are not sucked when the upper layer of plasma is collected; centrifuging the collected plasma again for 1000g and 10 min; after centrifugation is finished, sucking the upper layer of plasma until the remaining bottom liquid is 4% -5% of the blood sample volume to prepare the platelet-rich plasma, wherein the concentration of platelets is 1-2.5 multiplied by 10⁹In the embodiment, about 2ml of the PRP solution is left on the bottom of the tube, is evenly resuspended and counted, has PRP concentration which is about 4-5 times of the concentration of platelets in whole blood, meets the use requirement, does not contain red blood cells, and has even resuspension and no caking phenomenon. In other embodiments, the anticoagulant is dry powder directly coated on the inner wall of the anticoagulant tube or prepared into a solution with a concentration of 3.2-3.8% and coated on the inner wall of the anticoagulant tube for use after drying. The PRP concentration prepared by the invention is about 4-5 times of the platelet concentration in the whole blood, meets the use requirement, does not contain red blood cells, has even heavy suspension and no caking phenomenon,the calculated yield is more than 77%. The inventor finds in experiments that the anticoagulant can be prepared by using hydrogen-rich water, so that the phenomenon of PRP sticking and connecting blocks can be better avoided, and the suspension uniformity of the anticoagulant is ensured. Wherein the hydrogen-rich water is formed by adding zinc particles into purified water for reaction or formed by electrolyzing mineral water.

And mixing the activated adipose-derived stem cells with platelet-rich plasma to prepare a composite cell preparation, wherein the concentration of the activated adipose-derived stem cells is 4-6 million per milliliter, and the concentration of platelets is 3-5 million per milliliter. The final treatment scheme and cell preparation use unit and dosage, and a scheme conforming to the self-treatment is provided according to the actual condition and the physical examination report of the patient.

In use, as the parts of osteoarthritis generally have inflammation, and the inflammation needs to be eliminated quickly and effectively, a comprehensive treatment scheme is provided, namely, a certain amount of activated adipose-derived stem cells are injected into a joint cavity, and the activated adipose-derived stem cells have certain anti-inflammatory effect and efficacy. And then the treatment of the compound cell preparation of the activated stem cells and the PRP is carried out, the stem cells which are activated and begin to be transformed to the original chondrocytes quickly reach the surface of the wound and agglutinate to begin to differentiate by utilizing the homing effect of the stem cells, the activation induction time is saved, and the transformation rate is improved. PRP utilizes the anti-inflammatory action of the PRP and secretes more than 9 cytokines at the same time, promotes the transformation and the amplification procedure of stem cells, and under the combined action of 2, the PRP can quickly replace and repair local wound surfaces in an effective time, so that the optimal treatment effect is achieved.

We summoned 2 patients with gonarthritis, one of Wangzhi, women, 65 years old, suffered from gonarthritis for nearly 9 years, and suffered from pain and incapability of walking in severe cases. The other is that the president of an industry group is old, male is aged 64 years old, suffers from gonarthritis for 8 years, starts in autumn and winter every year, and becomes more serious along with the reduction of temperature. All 2 experimenters suffered from the pain of long-term knee arthritis, and the comprehensive physical examination of the experimenters met the experimental requirements of the people. The treatment plan is specially customized according to the actual physical condition of each person. After the knee joint cavity is locally injected with the novel cell preparation (activated adipose-derived stem cells + PRP) for 1 time per month after 3 months, tracking and recording are carried out, 2 patients all have a larger degree of improvement, Wangzuo walks easily and freely at present, pain is obviously relieved, and the effect is very obvious. The pain feeling is obviously relieved at present, the feeling of aggravation caused by the pain feeling along with the temperature reduction does not appear before, and the recovery is quite good. In addition, the treatment cost of the scheme is about 1/5 of the treatment cost of the original operation, so that the medical cost is greatly reduced, and the treatment cost of the gonitis is really reduced to two thousand yuan times.

While embodiments of the present invention have been described, the present invention is not limited to the above-described embodiments, which are intended to be illustrative rather than limiting, and many modifications may be made by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

1. A preparation method of a compound cell preparation applied to osteoarthritis is characterized by comprising the following steps:

2. The method of claim 1, wherein: the preparation method of the activated adipose-derived stem cells comprises the following steps:

3. The method of claim 2, wherein: the preparation steps of the adipose-derived stem cells are as follows:

4. The production method according to claim 3, characterized in that: after the adipose-derived stem cells grow to 80% -90% and fuse, the cells are cultured for five generations.

5. The production method according to claim 3, characterized in that: the cell activation culture medium is a serum-free lymphocyte culture medium added with interleukin-2 and saperin.

6. The method of claim 1, wherein: the preparation method of the platelet-rich plasma comprises the following steps:

7. The method of claim 6, wherein: the anticoagulant is citric acid, the prepared volume concentration is 0.109-0.129 mol/L, and the volume ratio of the anticoagulant to the blood sample is 1: 9.

8. the method of claim 6, wherein: the primary centrifugation is 200g and 8min, and the secondary centrifugation is 1000g and 10 min.

9. The method of claim 6, wherein: the anticoagulant is prepared by hydrogen-rich water.

10. The application method of the compound cell preparation in osteoarthritis is characterized by comprising the following steps: