阅读说明：本技术 一种在非变性情况下脱去生物体组织中细胞的方法 (Method for removing cells from organism tissue under non-denaturing condition ) 是由 宋云庆 姜来 于 2019-05-06 设计创作，主要内容包括：本发明公开了一种在非变性情况下脱去生物体组织中细胞的方法,将获取的生物体组织材料置于缓冲液内,加入核酸内切酶,25.0℃～40.0℃进行反应,反应过程缓慢加入盐溶液,并维持pH值在5～7之间。本发明所述的方法可以去除同种异体组织或异种异体组织中细胞及组织相容性抗原,且该方法在去除组织中细胞及组织相容性抗原的同时,最大程度的保留组织中对细胞及组织生长有益的生长因子。(The invention discloses a method for removing cells in organism tissues under a non-denaturing condition, which comprises the steps of placing an obtained organism tissue material into a buffer solution, adding endonuclease, reacting at 25.0-40.0 ℃, slowly adding a salt solution in the reaction process, and maintaining the pH value between 5 and 7. The method can remove the cell and tissue compatible antigens in the allogeneic tissues or the xenogeneic tissues, and can furthest retain growth factors which are beneficial to the growth of the cells and the tissues in the tissues while removing the cell and tissue compatible antigens in the tissues.)

1. A method for removing cells in organism tissues is characterized in that obtained organism tissue materials are placed in a buffer solution, endonuclease is added, reaction is carried out at 25.0-40.0 ℃, salt solution is slowly added in the reaction process, and the pH value is maintained to be 5-7.

2. The method of claim 1, wherein the biological tissue material:

(1) liquid tissue that is not dispersed and/or soluble in water, oil, or alcohol;

(2) excluding substantially non-cellular viable and dense tissue;

(3) excluding limbs containing blood vessels, nerves, muscles, skin.

3. The method according to claim 1, wherein the buffer is selected from one or more of sodium chloride, calcium chloride, lithium chloride, magnesium chloride and potassium chloride.

4. The method according to claim 3, wherein the total mass concentration of the buffer solution is 0.01-2.5%, and the preferred pH value is 4-6.

5. The method of claim 1, wherein the endonuclease is selected from the group consisting of a dnase and a rnase.

6. The method according to claim 5, wherein the concentration of the endonuclease is preferably 0.5 to 20U/μ L, more preferably 0.5 to 6U/μ L.

7. The process according to claim 1, wherein the temperature of the reaction is from 35.0 ℃ to 38.0 ℃.

8. The process according to claim 7, characterized in that the temperature difference varies preferably less than or equal to ± 0.1 ℃ during the same reaction.

9. The method of claim 1, wherein the salt solution is selected from the group consisting of: one or more of sodium chloride, calcium chloride, lithium chloride, magnesium chloride and potassium chloride.

10. The addition of the salt solution can be carried out in a titration mode, and the total mass concentration of the titrated solution is kept between 0.5 and 7 percent.

Technical Field

The invention relates to the technical field of biological medical treatment, in particular to a method for removing cells in organism tissues under a non-denaturing condition.

Background

The Non-destination decellularization technology is developed and derived based on the traditional preparation process of various common allogeneic products and xenogeneic products in the clinical use process at present. With the development of modern biomedical technology. We have found that human and animal tissue contains, in addition to a large number of cells and immunogenic substances, a large number of factors or proteins that are beneficial for wound repair, growth, induction, infection resistance, etc. Such as: human cortical growth factor (HEGP), osteoinductive growth factor (BMP), intravascular cortical growth factor (VEGF), and the like. These substances can induce or guide cells in the affected area for repair treatment of the damaged portion. At the same time, a plurality of factors and proteins can control the effect after tissue repair, and finally, the state which is almost the same as that before damage can be achieved. This is extremely important for repair of many injuries. Such as: skin grafting after burn, plastic cosmetology, etc.

For allogeneic and xenogeneic tissues, the problem of how to remove the immunogenicity carried on the tissue is first of all faced if it is to be applied to the patient's body. In the preparation process of traditional allogeneic and xenogeneic tissue products, the following steps are inevitably used: chemical reagents such as alcohols, ethers, ketones, strong oxides, strong acids, strong bases, etc.; or physically carrying out high temperature, high pressure, strong radiation and other processes. These methods can effectively remove immunogenic materials from tissues. But at the same time, various factors and proteins that are beneficial for healing of the affected area are almost completely destroyed. The product can only play a role of a pure biological scaffold after being used, and receptor cells can not be loaded and survive on the biological scaffold for a while. It finally has almost the same function as the biological scaffold made of many artificially synthesized materials. This greatly reduces the value as a biological tissue material.

Disclosure of Invention

The invention aims to provide a method for removing cells in organism tissues under a non-denaturing condition, which can remove cells and histocompatibility antigens in allogeneic tissues or xenogeneic tissues and maximally retain growth factors beneficial to the growth of the cells and the tissues in the tissues while removing the cells and the histocompatibility antigens in the tissues.

The tissue prepared by the method of the invention has the functions of the biological structure, the intercellular substance and part of the growth factors of the biological tissue material, does not retain the cell activity and does not acquire the functional products of the cell functionality, and is defined as a tissue engineering medical instrument (TEMP), namely: unlike the transplanted organ.

In order to achieve the purpose, the invention is realized by the following technical scheme:

a method for removing cells in organism tissue comprises the steps of placing an obtained organism tissue material in a buffer solution, adding endonuclease, reacting at 25.0-40.0 ℃, slowly adding a salt solution in the reaction process, and maintaining the pH value between 5 and 7.

The method of the present invention can continuously change the permeability of cells in the tissue through a specific pH value at a specific temperature, so that the reaction can be sufficiently performed.

In the present invention, the selection of the biological tissue material is an allogeneic tissue or a xenogeneic tissue commonly used in the art, and in the present invention, the following requirements are preferably applied:

(1) liquid tissues that are dispersed and/or soluble in water, oil, alcohol, such as: blood, lymph, cerebral medulla, etc.;

(2) tissues that are substantially non-cellular active and dense are excluded, such as: stratum corneum in the epithelium layer, hair tissue, nails, crowns, denser cortical bone in the skeleton, etc.;

(3) does not include complex and bulky body tissues or complex body tissues such as limbs containing blood vessels, nerves, muscles and skin. Examples include, but are not limited to, joints containing bone, cartilage, meniscus, tendon, and the like.

The material of the biological tissue of the present invention may be selected from allogeneic or xenogeneic tissues other than those mentioned above, including but not limited to skin, bone, cartilage, tendon, nerve tissue, blood vessels, heart valve, pericardium, amnion, umbilical cord tissue, etc.

The acquisition of the biological tissue material of the invention can follow the principle of obtaining the material of the allogeneic tissue material immediately after death of the donor, or refer to YY/T0513.1-2009 part 1 of allogeneic bone repair material: the method in basic requirements for bone tissue bank. The xenogenic tissue material can be obtained immediately after donor death. All material drawing work is preferably carried out in a controlled clean environment, so that the adhesion of microorganisms, heat sources and the like to the surface of the material during material taking is reduced. Meanwhile, the material should be taken out while avoiding touching or damaging the viscera (such as stomach, intestinal tract, etc.), so as to avoid serious pollution to the material.

If the materials are obtained, the preparation can be carried out immediately to be the best; if the preparation cannot be directly carried out, a method of cryopreservation can be employed. In general, direct cryogenic or cryogenic freezing may damage the material to some extent and may impair the use of the final tissue material. Preferably, the ice-free vitrification cryopreservation technology is used, so that the tissue material can be preserved for a long time and is not basically damaged.

The tissue after material drawing is not easy to be exposed in the air for a long time, thereby avoiding the dehydration and oxidation of the tissue material and avoiding irreversible damage to bioactive factors and the like carried in the tissue material. Preferably, physical isolation is selected for isolation, such as using: one or more of polyethylene film, paraffin, tinfoil or vacuum bag are isolated; or using a protective solution for isolation, such as: glycerol, normal saline, cotton seed oil, tween 80, monomethylsilicone oil, dimethyl sulfoxide or D-mannitol solution.

The biological tissue material obtained by the invention can be cut or cut into a required shape according to the requirements of a final product, the thickness and the layer number of the biological tissue material are limited as much as possible by preferably following the miniaturization principle when the size of the final product is prepared, the thickness of the biological tissue material is reduced as much as possible, for example, the preferable thickness is 0.01-6 mm, when good permeability can be ensured, the situations of folding, curling and the like are not easy to occur in the preparation process during operation. More preferably, the keratinized, dense and substantially inactive portion is further removed.

The biological tissue material obtained by the present invention is preferably prepared by preliminarily cleaning the surface of the tissue material with purified water or water in a clean environment. The purpose is to clean the residual blood, tissue fluid or protective solution on the surface of the tissue material and control the pollution of the tissue material by the pollution source in the environment. The cleaning time should not be too long, and the material should not be deformed by pressure and tensile force. After being taken out, no water drops, and drying can not be accelerated by using a drying and blowing mode.

Preferably, the biological tissue material after the preliminary cleaning is further cleaned by using a cleaning agent, wherein the cleaning agent is selected from one or more of polyoxyethylene alcohol, polysorbate, lauroyl sarcosinate, dimethyl sulfoxide, potassium laurate, methyl-p-phenylphenol or suramin sodium; the mass concentration of the cleaning agent is preferably between 0.01 and 45 percent, and the pH value is between 5.0 and 8.0; the cleaning time may be conventionally determined according to the characteristics of the material itself. It is preferable to uniformly wash the tissue material using an ultrasonic washer. After the washing is completed, the washing solution may be removed with an appropriate buffer. After the buffer solution is washed for 2-3 times, the tissue material can be immersed in the buffer solution.

The buffer according to the invention can be routinely determined according to the tissue material, for example, by selecting: one or more of sodium chloride, calcium chloride, lithium chloride, magnesium chloride and potassium chloride. The total mass concentration is preferably 0.01-2.5%, and the preferable pH is 4-6.

Furthermore, the endonuclease of the present invention can be one or more conventional broad-spectrum endonucleases, such as deoxyribonuclease (DNasel) and ribonuclease (RNase), to decompose nucleic acid and part of protein in cells in tissues, and the concentration is preferably 0.5 to 20U/. mu.L, more preferably 0.5 to 6U/. mu.L. Too high a concentration may result in unnecessary waste, while too low a concentration may not achieve the effect.

The reaction temperature of the invention is 25.0-40.0 ℃, preferably 35.0-38.0 ℃, and the temperature difference change is preferably less than or equal to +/-0.1 ℃ in the same reaction process; the specific temperature can be selected routinely according to the activation requirements of different endonucleases.

The reaction process of the invention needs to strictly maintain the pH value between 5 and 7, which is beneficial to increasing the permeability of cell membranes, and when the pH value is more than 7, the cell membranes and cell matrixes can be directly damaged.

The salt solution of the invention can be selected from: one or more of sodium chloride, calcium chloride, lithium chloride, magnesium chloride and potassium chloride in combination, and the concentration of the salt is not required to be specific, and the salt can be saturated solution of the salt.

The invention gradually changes the cell permeability of the organism tissue material by slowly adding the saline solution, and is beneficial to removing cells in the organism tissue.

The addition of the salt solution according to the invention can be carried out by conventional methods, for example by titration, and the total mass concentration of the salt in the titrated solution is kept between 0.5% and 7%.

The invention can use a stirrer or a shaking table to make the concentration of the buffer solution change uniformly while titrating.

According to the method, after the reaction is finished, the tissue material can be washed for 2-3 times by using the buffer solution.

The tissue material obtained after preparation can be sterilized by a Non-destination liquid sterilization method or other appropriate methods under the aseptic condition, and then one or more protective liquids are selected, such as: glycerin, dimethyl sulfoxide, polysorbate, polyoxyethylene alcohol, acetamide and the like to protect tissue materials; the tissue material is preserved in a sealing mode, and can be preserved for a long time by using an ice-free vitrification freezing and preserving technology or other appropriate modes.

The method of the invention has the following advantages over the prior art:

the method of the invention is completely different from the traditional preparation process of biological tissue products, and adopts salt solution and endonuclease to treat the biological tissue under specific environment. The technology can be used for treating the biological tissue, so that the tissue can remove unwanted cells and immunogenicity, and simultaneously, the cells, active ingredients and the like which are beneficial to the growth and healing of the affected part in the biological tissue are furthest reserved. A large number of experiments show that the tissue treated by the technology not only can perfectly induce or guide the receptor cells to quickly repair the damaged part, but also is an excellent stem cell carrier, so that the receptor stem cells can quickly colonize and survive in the tissue, differentiate and participate in tissue repair.

Drawings

Fig. 1 is a photomicrograph of the allogeneic dermal matrix prepared in example 1;

fig. 2 is a photomicrograph of the allogeneic dermal matrix prepared in comparative example 1;

FIG. 3 shows the adherent growth of a basal lamina on the dermal matrix prepared in example 1;

fig. 4 shows the adhesion growth of a basal lamina on the dermal matrix prepared in comparative example 1.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.

The method of the following embodiments may involve an apparatus comprising: a precise constant temperature water domain device, a titrator, an ultrasonic cleaning machine, a shaking table, a vacuum packaging machine and the like.

In the sampling of xenogenic tissue material, a standardized animal breeding facility was selected which should provide animal attributes and three generations of ancestry evidence, as well as investigation and elimination of animals likely to contain Transmissible Spongiform Encephalopathy (TSE) factors. Reference may be made to YY/T0771.4-2015 "part 4 of animal derived medical devices: removal and/or inactivation of Transmissible Spongiform Encephalopathy (TSE) factors and their course confirm the principles of the assay.

In the following examples, the titration of tissue material, the determination of reaction time and the detection of residual cells can be performed according to the guideline for testing the activity of biomaterial scaffold in tissue engineering medical device products, which is referenced by YY/T1562-2017, or the methods commonly used in the art, such as allogeneic tissue material and xenogeneic tissue material, should be prepared to the specified specifications according to the actual product requirements. The slice is mainly thin, the thickness is preferably 0.1-6 mm, and thinner slices can be prepared. The appropriate buffer and endonuclease are selected and the above preparation is repeated at the appropriate pH and temperature. Selecting a sampling time point according to the type and characteristics of the tissue material: such as: 0.1 h/time, 0.5 h/time, 1 h/time, 3 h/time, etc. (if the tissue material thickness is less than 0.1mm, it is recommended to shorten the sampling time point appropriately), tissue sectioning is performed. Selecting appropriate coloring agent, such as: hematoxylin, carmine, aniline blue, brilliant green, eosin, basic fuchsin, methylene blue, methyl green, etc., and staining of tissue sections and observation of hydrolysis of cellular chromosomes in the tissue. When no stained chromosomes are observed, or no viable cells are detected. The sampling time point of the sample should be set as the titration and reaction time of the tissue material (in the mass production process, the titration and reaction time is usually increased by 5% -20% to ensure the safety and final qualified rate of the final product, but the sampling time point should be specifically set according to the characteristics of the tissue material). The above method can also be used in the form of a final examination of the tissue material.

In the following examples, the prepared allogeneic tissue material and xenogeneic tissue material will be used clinically, and the immunogenic substances in the tissue material must be strictly controlled. The immunogenicity of the tissue material may be determined and evaluated by methods described in YY/T1465.1-2016, section 1 of methods for evaluating immunogenicity in medical devices, in vitro T lymphocyte transformation assays, or by methods commonly used in the art, such as in vitro lymphocyte stimulation assays, to quickly determine whether the tissue material retains immunogenic substances. The specific operation can be as follows: taking a proper amount of tissue material samples and leaching liquor, operating under an aseptic condition, smashing the samples and mixing with the leaching liquor at 37 ℃, standing for 12-72 h, centrifuging at 3000-6000 rpm for 3-10 min, taking supernatant leaching liquor, and freezing and storing at-20 ℃. Separating lymphocytes in peripheral blood of healthy adult by using lymphocyte separation liquid, preparing into suspension, and adjusting concentration to 1-3 x 10⁶And/ml. According to the MTT cell proliferation assay, 96-well plates were used in three groups: a. a stimulation group; 50-100 mul of human lymphocyte and leaching liquor respectively; b. positive control group: 50-100 mul of human lymphocyte and PHA (phytohemagglutinin) respectively; c. negative control group: man showerThe average value of 50-100 mul each of the Bara cells and the blank buffer solution was taken. And (3) placing the mixture in a 5% carbon dioxide incubator at 37 ℃ for 24-72 h, observing the growth condition of the cells every day, centrifuging, taking supernate, adding DMSO (100-150 mu l/hole), and carrying out shake reaction for 5-10 s. Absorbance (A) was measured at a certain wavelength on a microplate reader, and the average of absorbance A for each group was recorded and compared. If the tissue material does not contain immunogenic substances, the absorbance comparison should be: the test group is less than or equal to the negative control group and less than the positive control group.