CN110338188B

CN110338188B - Neuron cryopreservation liquid and neuron cryopreservation and recovery method

Info

Publication number: CN110338188B
Application number: CN201910516679.2A
Authority: CN
Inventors: 郭安臣; 王群; 王拥军
Original assignee: Beijing Tiantan Hospital
Current assignee: Beijing Tiantan Hospital
Priority date: 2019-06-14
Filing date: 2019-06-14
Publication date: 2021-08-31
Anticipated expiration: 2039-06-14
Also published as: CN110338188A

Abstract

The invention discloses a neuron cryopreservation solution which comprises the following components in parts by volume: 45.00-55.00 parts of neuron culture medium, 40.50-49.50 parts of 10% bovine serum albumin and 4.50-5.50 parts of dimethyl sulfoxide; the neuron frozen stock solution does not contain glutamine. Above-mentioned neuron cryopreservation liquid, bovine serum albumin can protect neuron cells, can alleviate the damage of adverse environment to neuron cells, can utilize dimethyl sulfoxide's effect to make the solution freezing point reduce, and under the slow freezing condition, intracellular moisture permeates out, has reduced the ice crystal formation to avoid cell damage, can avoid dimethyl sulfoxide concentration too high again, produce serious toxic action to freezing cell, lead to protein degeneration. In addition, the neuron cryopreservation solution does not contain glutamine which is generally added in a common culture medium, so that the neuron cryopreservation solution has less cytotoxicity compared with a freezing solution containing glutamine, and the neuron activity after recovery is higher.

Description

Neuron cryopreservation liquid and neuron cryopreservation and recovery method

Technical Field

The invention belongs to the technical field of tissue cell culture, and particularly relates to a neuron cryopreservation solution and a neuron cryopreservation and resuscitation method.

Background

In studies around the nervous system, primary isolation culture of neurons is a very important in vitro study for studying various physiological functions of nerve cells, and is mainly performed on rodent mammals with respect to primary isolation of neurons due to limitations obtained by nerve tissues. For example, the rodents that are commonly used for primary isolation of neurons are fetal mice, neonatal mice. However, since the neuron cells lack the proliferation ability, primary separation is not suitable for long-term survival, growth and differentiation in vitro, which is not favorable for the smooth research; in addition, for primary neurons, the neurons are particularly sensitive to the external environment and are extremely easy to die after being badly stimulated, so in vitro experiments of primary cells of nerve tissues, fresh tissue culture is currently separated, and the research period cost is increased comprehensively due to the limitation of material obtaining of research targets, such as difficulty in obtaining fetal mice and newborn mice and long period, when the primary neurons are researched.

Cell freezing technology has been extensively used in the field of biology as an effective method for preserving cells. Researchers often need to freeze cells and store them for later experimental or clinical use. The traditional low-temperature cryopreservation technology is to add dimethyl sulfoxide and serum into a cell suspension, then place the cell suspension into a cryopreservation tube, slowly freeze the cell suspension in a refrigerator at the temperature of minus 80 ℃, place the cryopreservation tube in liquid nitrogen for preservation, enable cells to be temporarily separated from a growth state and preserve the cell characteristics, and rapidly thaw the cells when needed. Moreover, a certain amount of cells are preserved appropriately, so that the cells can be prevented from being lost due to contamination or other accidents of the cultured cells, and the function of preserving the cells is achieved. In addition, some cells can be purchased, hosted, exchanged, and transported in a frozen form. When the cells are frozen, glycerol or dimethyl sulfoxide (DMSO) with the final concentration of 5% -15% of a protective agent is added, so that the freezing point of the solution can be lowered, water in the cells can permeate out under the condition of slow freezing, and the formation of ice crystals is reduced, thereby avoiding cell damage. The method of 'slow freezing and fast melting' can better ensure the survival of the cells.

If the primary neurons are cryopreserved by adopting a cryopreservation technology, the separation cost can be solved, and sample materials can be used to the maximum extent. However, when primary neurons are frozen by using a dimethyl sulfoxide and serum mixed solution as a freezing solution in the prior art, freezing damage is often caused to the neurons, so that the neurons can not continue to normally survive after recovery. The damage to cells caused by low-temperature cryopreservation mainly comprises the following aspects: the fluidity of cells is reduced, cell membrane speech causes further damage to cells, and Na is inhibited⁺/K⁺Atpase, causing cell swelling; leading to necrotic apoptosis of cells; oxygen radicals are generated. One of the key factors in reducing damage to primary neurons caused by low-temperature cryopreservation is to provide a suitable cryopreservation solution.

Disclosure of Invention

The invention provides a neuron cryopreservation solution and a neuron cryopreservation and recovery method, aiming at the problems of low neuron survival rate and poor neuron survival rate and activity caused by the fact that the traditional cryopreservation solution cannot avoid neuron cryopreservation injury.

The invention provides a neuron cryopreservation solution which comprises the following components in parts by volume: 45.00-55.00 parts of neuron culture medium, 40.50-49.50 parts of 10% bovine serum albumin and 4.50-5.50 parts of dimethyl sulfoxide; the neuron cryopreservation solution does not contain glutamine.

In one embodiment, the neuron culture medium comprises the following components in parts by volume: 45.00-55.00 parts of Neurobasal A culture medium or Neurobasal culture medium and 0.90-1.10 parts of B27 cell culture additive.

In one embodiment, the neuron culture medium further comprises the following components in parts by volume: 0.27 to 0.33 portions of double antibody.

In one embodiment, the Neurobasal a medium is glycoform.

The invention also discloses a neuron cryopreservation and recovery method, which comprises the following steps:

sampling: taking nerve tissue of an isolated mammal, cutting into pieces, placing the cut pieces in a first centrifuge tube, centrifuging for 4-6 min at 800-1200 rpm, and removing a centrifugation supernatant;

digestion: adding pancreatin into the first centrifuge tube, blowing off the precipitated cells in the first centrifuge tube, pumping out the precipitated cells, placing the precipitated cells into a digestion culture dish, adding pancreatin into the digestion culture dish, placing the digestion culture dish into an incubator at 37 ℃ for 8-12 min, and adding a planting culture medium into the digestion culture dish to terminate digestion;

preparing a cell suspension: filtering the mixture in the digestion culture dish by using a cell sieve, centrifuging at 800-1200 rpm for 4-6 min, and removing supernatant after centrifugation to prepare primary neurons;

freezing and storing: mixing the primary neurons with the cryopreservation solution of any one of claims 1 to 5 to prepare a cryopreserved cell sap, placing the cryopreserved cell sap in a cryopreservation tube, freezing the cryopreserved cell sap in a refrigerator at the temperature of-20 ℃ for 1.0 to 2.0 hours, and freezing the cryopreserved cell sap in a refrigerator at the temperature of-80 ℃;

and (3) resuscitation: and quickly placing the cryopreserved tube in a water bath at 37 ℃ for instant dissolution, centrifuging the thawed cryopreserved cell sap at 800-1200 rpm for 4-6 min, removing a supernatant after centrifugation to prepare cryopreserved neurons, and transferring the cryopreserved neurons into a recovery culture medium for culture or a planting culture medium for culture.

In one embodiment, before the freezing step, the method further comprises the following steps: and mixing the primary neurons with the planting culture medium, centrifuging at 800-1200 rpm for 4-6 min, and removing a supernatant after centrifuging.

In one embodiment, in the recovering step, before transferring the cryopreserved neurons into a recovery medium culture or a planting medium culture, the method further comprises the following steps: mixing the frozen neurons with a resuscitation medium, centrifuging at 800-1200 rpm for 4-6 min, and removing a supernatant after centrifugation.

In one embodiment, the neural tissue includes any one of hippocampal tissue, cerebral cortex, and brain tissue.

In one embodiment, the planting medium comprises the following components in parts by volume: 36.53-45.87 parts of DMEM medium, 4.50-5.50 parts of horse serum and 2.70-3.30 parts of fetal bovine serum.

In one embodiment, in the recovery step, the neurons after being frozen are transferred to a planting medium for culture for 4 hours and then are replaced by a neuron medium for culture.

Above-mentioned neuron cryopreservation liquid, including neuron culture medium, bovine serum albumin and dimethyl sulfoxide, bovine serum albumin can protect neuron cells, can alleviate adverse environment to neuron cell's damage, can utilize dimethyl sulfoxide's effect to make the solution freezing point reduce, and under the slow freezing condition, intracellular moisture permeates out, has reduced the ice crystal formation to avoid cell damage, can avoid dimethyl sulfoxide concentration too high again, produce serious toxic action to freezing cell, lead to protein denaturation. In addition, the neuron cryopreservation solution does not contain glutamine which is generally added in a common culture medium, so that the neuron cryopreservation solution has less cytotoxicity compared with a freezing solution containing glutamine, and the neuron activity after recovery is higher.

Drawings

FIGS. 1 to 4 are graphs showing the results of immunofluorescence staining according to example 1 of the present invention;

FIGS. 5 to 8 are graphs showing the results of immunofluorescence staining according to example 2 of the present invention;

FIG. 9 is a graph showing the results of immunofluorescence staining of comparative example 1 of the present invention;

FIG. 10 is a graph showing the results of immunofluorescence staining of comparative example 2 of the present invention;

FIG. 11 is a graph showing the results of immunofluorescence staining of comparative example 3 of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the detailed description and specific examples, while indicating the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.

The neuron cryopreservation liquid provided by the invention comprises the following components in parts by volume: 45.00-55.00 parts of neuron culture medium, 40.50-49.50 parts of 10% bovine serum albumin and 4.50-5.50 parts of dimethyl sulfoxide; the neuron frozen stock solution does not contain glutamine.

Wherein Bovine Serum Albumin (BSA), CAS number 9048-46-8, also known as the fifth component, is an Albumin in bovine serum, contains 583 amino acid residues, has a molecular weight of 66.430kDa, and has an isoelectric point of 4.7. In the cryopreservation solution disclosed by the invention, BSA (bovine serum albumin) can protect neuronal cells, can reduce damage of adverse environment to the neuronal cells, can lower the freezing point of the solution by utilizing the action of dimethyl sulfoxide, can permeate water in the cells under a slow freezing condition, and can reduce the formation of ice crystals, thereby avoiding cell damage, and can also avoid serious toxic effect on the cryopreserved cells due to overhigh concentration of dimethyl sulfoxide to cause protein denaturation. Furthermore, the neuron cryopreservation solution does not contain glutamine which is commonly added in a common culture medium, so that the neuron cryopreservation solution has lower cytotoxicity compared with a solution containing glutamine, and the neuron activity after recovery is higher. Alternatively, 10% BSA is an aqueous solution with a BSA concentration of 10% by mass.

Preferably, the neuron cryopreservation solution comprises the following components in parts by volume: 49.00-51.00 parts of neuron culture medium, 44.50-45.50 parts of 10% BSA and 4.90-5.10 parts of dimethyl sulfoxide. More preferably, the neuron cryopreservation solution comprises the following components in parts by volume: 50.00 parts of neuron culture medium, 45.00 parts of 10% BSA, and 5.00 parts of dimethyl sulfoxide. The research shows that the components of the neuron freezing medium cooperate with each other to play a role in synergy, and if the content of a certain component is too high or too low, the death or activity of neurons can be reduced. For example, when the content of dimethyl sulfoxide is too high, the toxicity of cells is too high to cause neuronal death. As another example, the volume ratio of neuronal medium to 10% bovine serum albumin is in the range of 1: (0.85-0.95) is preferable because bovine serum albumin can protect neurons from damage, but when it exists alone, it does not have the condition for protecting or maintaining the survival of neurons, so that the ratio of the neuron culture medium and the bovine serum albumin needs to be properly adjusted to coordinate the action between the two, thereby providing the environment most favorable for neurons.

As an alternative embodiment, the neuron culture medium comprises the following components in parts by volume: 45.00-55.00 parts of Neurobasal A or Neurobasal culture medium and 0.90-1.10 parts of B27 cell culture additive. Wherein, Neurobasal A culture medium is used for culturing newborn and adult neurons, Neurobasal A culture medium is used for culturing prenatal and fetal neurons, and Neurobasal A culture medium and Neurobasal culture medium do not contain L-glutamine, L-glutamic acid or aspartic acid. The corresponding Neurobasal A medium or Neurobasal medium can be selected according to the source of the neuron. B27 cell culture additive is a serum-free cell culture additive most recently developed optimally by englen biology (Engreen biosystems co., Ltd) according to relevant cell culture characteristics.

Preferably, the neuron culture medium comprises the following components in parts by volume: 45.00-55.00 parts of Neurobasal A or Neurobasal culture medium, 0.90-1.10 parts of B27 cell culture additive and 0.27-0.33 part of double-antibody. The double antibody is a Streptomycin mixed Solution, and the Streptomycin mixed Solution (100X) (Penicilin-Streptomycin Solution) double antibody is specially used for cell culture and can be directly added into a cell culture Solution. In the penicillin-streptomycin solution (100X), the content of penicillin is 10000U/mL, and the content of streptomycin is 10 mg/mL. The preferred working concentration of penicillin in the cell culture broth is 100U/mL, the working concentration of streptomycin is 0.1mg/mL, and the final concentration of penicillin is 100 units/mL. The addition of double antibody can inhibit the growth of bacteria and avoid cell pollution. When in use, the double-resistant can be configured according to needs, or the finished product of the double-resistant is purchased on the market. More preferably, the neuron culture medium comprises the following components in parts by volume: 50.00 parts of sugar type Neurobasal A culture medium, 1.00 part of B27 cell culture additive and 0.30 part of double antibody.

As an alternative embodiment, the neuron culture medium in the frozen stock solution of the invention adopts Neurobasal A culture medium, and the Neurobasal A culture medium is a glycoform.

The invention also provides a neuron cryopreservation and resuscitation method, which comprises the following steps:

digestion: adding pancreatin into the first centrifuge tube, blowing off precipitated cells in the first centrifuge tube, pumping out the precipitated cells, placing the precipitated cells into a digestion culture dish, adding pancreatin into the digestion culture dish, placing the digestion culture dish into an incubator at 37 ℃ for 8-12 min, and then adding a planting culture medium into the digestion culture dish to terminate digestion;

freezing and storing: mixing primary neurons with the cryopreservation solution to prepare a cryopreservation cell sap, placing the cryopreservation cell sap in a cryopreservation tube, freezing the cell sap in a refrigerator at the temperature of-20 ℃ for 1.0 to 2.0 hours, and then freezing the cell sap in the refrigerator at the temperature of-80 ℃;

and (3) resuscitation: and (3) quickly placing the cryopreserved tube in a water bath at 37 ℃ for instant dissolution, centrifuging the thawed cryopreserved cell sap at 800-1200 rpm for 4-6 min, removing supernatant after centrifugation to prepare cryopreserved neurons, and transferring the cryopreserved neurons into a recovery culture medium for culture or a planting culture medium for culture.

According to the neuron cryopreservation and recovery method, the primary neurons and the cryopreservation liquid are mixed and then cryopreserved, so that neuron cryopreservation damage can be reduced, and the recovered neurons are high in survival rate and strong in activity. Furthermore, the method for sampling and separating the nerve tissue is quick and convenient, avoids neuron damage and cell pollution, improves sampling efficiency and is beneficial to subsequent digestion treatment.

Specifically, in the resuscitation step, the culture medium used for resuscitation may be selected according to the purpose of resuscitation, i.e., the resuscitation culture medium may be selected for resuscitation culture or the planting culture medium may be selected for resuscitation culture according to the purpose of neuron culture. When the recovery culture medium is selected for recovery culture, a product containing neurons and astrocytes can be recovered and cultured, and the total amount of positive cells reaches more than 70%; when the planting medium is selected for recovery culture, a product containing neurons can be recovered and cultured, and the number of positive cells of the neurons reaches more than 90%.

As an alternative embodiment, before the step of freezing, the method further comprises the following steps: mixing the primary neurons with a planting culture medium, centrifuging at 800-1200 rpm for 4-6 min, and removing a supernatant after centrifuging. The planting medium is mixed with the primary neurons before freezing and then centrifuged, which is beneficial to purifying the primary neurons and avoiding the damage of the neurons caused by the influence of substances such as pancreatic enzymes and the like remained in the digestion step on the environment during freezing or recovery.

As an alternative embodiment, in the resuscitation step, before transferring the cryopreserved neurons into the resuscitation medium for culture, the method further comprises the following steps: mixing the frozen neurons with a resuscitation medium, centrifuging at 800-1200 rpm for 4-6 min, and removing a supernatant after centrifugation. The planting medium and the primary neurons are mixed and then centrifuged before recovery, which is beneficial to purifying the primary neurons and avoiding the damage of the neurons caused by the influence of residual frozen stock solution on the recovery environment.

Optionally, the neural tissue includes any one of hippocampal tissue, cerebral cortex, and brain tissue.

As an alternative embodiment, the planting medium comprises the following components in parts by volume: 36.53-45.87 parts of DMEM medium, 4.50-5.50 parts of horse serum and 2.70-3.30 parts of fetal bovine serum. Wherein, DMEM (Dulbecco's modified eagle medium) is a culture medium containing various amino acids and glucose, and the DMEM culture medium selected by the invention is a sugar-containing culture medium without L-glutamine. Horse Serum (HS) and Fetal Bovine Serum (FBS) used in the planting medium are commonly used cell culture additives. Although DMEM medium is a common cell culture medium, the research of the invention unexpectedly finds that DMEM medium, HS and FBS can be combined to obtain better culture effect as a planting medium, and the neuron cultured by the planting medium prepared by the components in a ratio has stronger activity.

Preferably, the planting medium comprises the following components in parts by volume: 40.00-43.40 parts of DMEM medium, 4.90-5.10 parts of HS and 2.90-3.10 parts of FBS. More preferably, the planting medium comprises the following components in parts by volume: 41.70 parts DMEM medium, 5.00 parts HS, 3.00 parts FBS and 0.30 parts double antibody.

As an alternative embodiment, in the recovery step, the neurons after being frozen are transferred to a planting medium for culture for 4 hours and then are replaced by a neuron medium for culture.

As an alternative embodiment, the optional resuscitation medium provided by the present invention comprises the following components in parts by volume: 45.00-55.00 parts of neuron culture medium, 45.00-55.00 parts of neural stem cell culture medium, 0.90-1.10 parts of 10% BSA and 0.90-1.10 parts of 100 Xnerve growth factor. Among them, Nerve Growth Factor (NGF) is a protein that can regulate the growth and development of peripheral and central neurons, maintain the survival of neurons, and has dual biological functions of neuron nutrition and neurite growth promotion.

Preferably, the resuscitation medium comprises the following components in parts by volume: 49.00-51.00 parts of neuron culture medium, 49.00-51.00 parts of neural stem cell culture medium, 0.95-1.05 parts of 10% BSA and 0.95-1.05 parts of 100 XNGF. More preferably, the resuscitation medium comprises the following components in parts by volume: 50.00 parts of neuron culture medium, 50.00 parts of neural stem cell culture medium, 1.0 part of 10% BSA, and 1.0 part of 100 XNGF.

As an alternative embodiment, the neural stem cell culture medium comprisesThe following components in parts by volume: 41.50-49.50 parts of DMEM/F12 culture medium, 0.90-1.10 parts of basic fibroblast growth factor, 0.90-1.10 parts of epidermal growth factor, 0.90-1.10 parts of B27 cell culture additive, 0.45-0.55 part of N2 cell culture additive and 0.45-0.55 part of double antibody. The basic fibroblast growth factor (bFGF) belongs to an FGF family, is a mitogenic cationic polypeptide containing 155 amino acids, has the molecular weight of 16-18.5 KD, can promote the proliferation of a series of cells including mesenchymal cells, neuroectoderm and vascular endothelial cells, and has potential angiogenesis promoting activity in vivo. In addition, bFGF is an important component of embryonic stem cell culture medium, which maintains cells in an undifferentiated state in serum-free medium. bFGF is a neurotrophic factor, a mitogen for glial cells and schwann cells. bFGF has non-mitotic activity to stimulate glial cells, such as promoting astrocyte migration and the release of plasmin activators; regulating the expression of Glial Fibrillary Acidic Protein (GFAP) and the synthesis of glutamic acid and S-100 protein; changes in the cellular processes and cell membrane structures typical of astrocytes; promoting proliferation of astrocytes and forming a fibrous appearance; it also can promote oligodendrocyte proliferation and increase myelin-associated protein and lipid content. In vitro neuronal cultures, bFGF prolongs the survival of a variety of central and peripheral neurons in culture, stimulates the synthesis of choline acetylase and the growth of processes. It has been reported that addition of bFGF to cultured hippocampal neurons of fetal rats increases the survival time of the neurons and their axons. Adding 10-30 pg/mL bFGF to cultured hippocampal neurons of fetal rats^-1The life of the neuron which can only survive for 5-7 days is prolonged by 14 days, and the number of the neuron is increased by 4 times; when the concentration is increased to 200-500 pg/mL^-1In this case, the protrusions having a size of only 30 μm can be extended to 100 μm. After bFGF is added to cultured Schwann cells, 5-10% of the cells enter the division phase. bFGF is applied to cholinergic neuron and dopaminergic neuron of frontal region, apical region, striatum and thalamus of embryonic mouse brain, rat cerebellar cortical neuron, sympathetic ganglion cell and chick embryo spinal cord anterior horn neuron in cultureEtc. have nourishing and promoting effects. bFGF also has a proliferative and differentiating effect on neural precursor cells. It was found that upon addition of bFGF to cultured rat neurons, cholinergic components are cleaved and proliferated. It has also been observed that the division of neuroblasts is regulated by bFGF, with the development of neurite outgrowth from the growth cone, neurotransmitter synthesis, transport of transmitter vesicles, and other neuronal properties occurring during the division. In addition, bFGF may also affect the development of the central and peripheral nervous system through its pro-angiogenic effect. Epidermal Growth Factor (EGF), also known as human oligopeptide-1, is an active substance in human body, and an active polypeptide consisting of 53 amino groups can repair and proliferate skin surface cells by stimulating tyrosine phosphorylation of EGF receptor. N2 cell culture additive is a serum-free cell culture additive most recently developed optimally according to relevant cell culture characteristics by englen biosystems co.

Preferably, the neural stem cell culture medium comprises the following components in parts by volume: 44.50-45.50 parts of DMEM/F12 culture medium, 0.95-1.05 parts of bFGF, 0.95-1.05 parts of EGF, 0.95-1.05 parts of B27 cell culture additive, 0.48-0.52 parts of N2 cell culture additive and 0.48-0.52 parts of double antibody.

Preferably, the neural stem cell culture medium comprises the following components in parts by volume: 45.00 parts of DMEM/F12, 1.00 parts of bFGF, 1.00 parts of EGF, 1.00 parts of B27 cell culture additive, 0.50 parts of N2 cell culture additive and 0.50 parts of double antibody.

For the sake of convenience of illustration and comparison, in the following examples and comparative examples of the present invention, the cryopreservation solution and the resuscitation medium used may be further prepared based on the following ratios of the planting medium, the neuron medium and the neural stem cell medium. It should be noted that, for comparison, the following planting medium, neuron medium and neural stem cell medium all adopt the better ratio of the ratios, and under the condition of other ratios of the medium of the present invention, a large number of experiments have been performed to prove that the purpose of the present invention can be achieved, and the comparison of the effects of other ratios is not repeated herein.

Example 1

(1) Preparation of the experiment: coating 4 24-pore plates and 2 6-pore plates with polylysine, removing coating liquid after overnight at 37 ℃, and blowing for 2h until drying; after 2 times of aseptic water washing, adding 0.3 mL/hole of glycoform DMEM culture medium, extracting the DMEM culture medium before cell planting, replacing a 24-hole plate with 500-mu L planting culture medium, and replacing a 6-hole plate with 2mL planting culture medium; sterilizing surgical instruments including scissors, tweezers and fiber tweezers.

(3) And (5) carrying out sterilization microscopy and anesthesia on the animal to be tested, namely the 5-day embryonic mouse.

(4) Sampling: cutting the skull of an embryonic mouse, taking out brain tissue, stripping blood vessels and meninges in ice-cold DMEM medium, cutting the brain tissue, drawing out, placing the brain tissue in a 15mL first centrifuge tube, centrifuging at 1000rpm for 5min, and removing the centrifugal supernatant.

(5) Digestion: adding 6.0mL of 0.25% pancreatin into the first centrifuge tube, blowing off the precipitated cells in the first centrifuge tube, extracting the precipitated cells, placing the precipitated cells into a digestion culture dish, adding 1.0mL of 0.25% pancreatin into the digestion culture dish, placing the digestion culture dish into an incubator at 37 ℃ for 10min, and adding 2mL of planting culture into the digestion culture dish to terminate digestion.

(6) Preparing a cell suspension: the mixture in the digestion culture dish is extracted and placed in a 15mL second centrifuge tube, at the moment, the cells in the digestion culture dish are pasty, extraction and blowing are carried out simultaneously, and the cells are blown away as much as possible; and (3) screening the cells in the second centrifuge tube through a cell screen, adding 2mL of planting medium into the second centrifuge tube added with the cells to wash away the residual cells in the second centrifuge tube and screen the cells, pumping the cells obtained through twice cell screening back into the centrifuge tube, centrifuging at 1000rpm for 5min, and removing the supernatant after centrifugation to obtain the primary neuron.

(7) Planting: adding 5.00mL of planting medium into a centrifuge tube for preparing primary neurons, blowing for about 30 times until no cells agglomerate, planting 500 mu L of planting medium containing cells into a 24-well plate processed in the experiment preparation step, and planting 1.00mL of planting medium containing cells into a 6-well plate processed in the experiment preparation step; after 4h, the neuron culture medium is replaced by a half-amount liquid replacement mode.

Freezing and storing: mixing the prepared primary neurons with 10mL of cryopreservation liquid to prepare cryopreservation cell sap, placing the cryopreservation cell sap in a cryopreservation tube, freezing for 1.5h in a refrigerator at the temperature of-20 ℃, and freezing in a refrigerator at the temperature of-80 ℃.

(8) And (3) resuscitation: quickly placing the frozen tube in a water bath at 37 ℃ for instant dissolution, centrifuging the frozen cell sap after melting at 1000rpm for 5min, removing supernatant after centrifugation to obtain neurons after freezing, transferring the neurons after freezing into a planting culture medium for culture for 4h, and then replacing the neurons with a neuron culture medium for culture.

(9) Immunofluorescence staining experiment: the recovered neurons were subjected to immunofluorescence staining assay (primary antibody: Tub-M1: 200GFAP-R1:200 secondary antibody: GM488GR 5681: 500) and the results are shown in FIGS. 1 to 4.

The mixture ratio of the planting culture medium, the neuron culture medium and the freezing medium adopted in the embodiment 1 is as shown in the specification:

the volume ratio of the components of the planting culture medium is as follows: 41.70mL DMEM, 5.00mL HS, 3.00mL FBS, 0.30mL double antibody.

The neuron culture medium comprises the following components in percentage by volume: 50.00mL Neurobasal A, 1.00mL B27, 0.30mL diabody.

The frozen stock solution comprises the following components in percentage by volume: 50.00mL of neuronal medium, 45.00mL of 10% bovine serum albumin, and 5.00mL of dimethyl sulfoxide.

As shown in fig. 1 to fig. 4, it can be estimated that: Tub/DAPI is 14/15 ≈ 90%, GFAP/DAPI is 0/15 ≈ 0%, Tub positive cell count accounts for 90%, and GFAP positive cell count accounts for 0%. From the calculation results, it is understood that the frozen stock solution prepared in example 1 has a high neuron cell survival rate after frozen stock, and from fig. 2, the neuron cells cultured by resuscitation are round and have thick and long protruding processes, indicating that the neuron activity is strong. Experiments prove that after the special neuron culture medium is used for culturing for 4 hours, the special neuron culture medium is continuously cultured, only target neurons are recovered, astrocytes are avoided, the neurons can be conveniently and independently researched, the phenomenon of apoptosis gradually occurs after 20 days of the cultured neurons are recovered, and the in-vitro survival life of the neurons is greatly prolonged.

Example 2

Example 2 differs from example 1 in that in the recovery step, the cryopreserved neurons were transferred to recovery medium for culture. The mixture ratio of the planting culture medium, the neuron culture medium, the neural stem cell culture medium, the freezing medium and the resuscitation culture medium adopted in the example 2 is as follows:

The volume ratio of the components of the neural stem cell culture medium is as follows: 45.00mL DMEM/F12, 1.00mLbFGF, 1.00mLEGF, 1.00mLB27, 0.50mLN2, 0.50mL double antibody.

The volume ratio of the components of the resuscitation culture medium is as follows: 50.00mL of neuronal medium, 50.00mL of neural stem cell medium, 1.0mL of 10% BSA, and 1.0mL of 100 XNGF.

The neurons recovered in example 2 were subjected to immunofluorescence staining (primary antibody: Tub-M1: 200GFAP-R1:200 secondary antibody: GM488GR 5681: 500), and the results are shown in FIGS. 5 to 8, and it was determined that: Tub/DAPI is 15/138 ≈ 11%, GFAP/DAPI is 81/138 ≈ 59%, Tub-positive cells account for 11%, and GFAP-positive cells account for 59%. From the above calculation results, the total cell survival rate after cryopreservation of the cryopreservation solution prepared in example 2 is high, and as can be seen from fig. 6 and 7, the neuron cells obtained by recovery culture are round and smooth, and the protruding processes are thick and long, which indicates that the neuron activity is strong, and a large number of astrocytes also survive, thereby facilitating the study of the interaction between astrocytes and neurons. Experiments prove that the phenomenon of apoptosis gradually appears after the neurons and the astrocytes are recovered and cultured for 20 days, and the survival time of the neurons and the astrocytes in vitro is prolonged.

Example 3

Example 3 differs from example 1 in that in the recovery step, the cryopreserved neurons were transferred to a planting medium for culture. The mixture ratio of the planting culture medium, the neuron culture medium, the neural stem cell culture medium, the freezing medium and the resuscitation culture medium adopted in the embodiment 3 is as follows:

The frozen stock solution comprises the following components in percentage by volume: 45.00mL of neuronal medium, 45.00mL of 10% bovine serum albumin, 4.50mL of dimethyl sulfoxide.

The neurons recovered in example 3 were subjected to immunofluorescence staining test (primary antibody: Tub-M1: 200GFAP-R1:200 secondary antibody: GM488GR 5681: 500), and the number of Tub-positive cells and the number of GFAP-positive cells were calculated to be 92% and 0%, respectively. The neuron cells cultured by resuscitation are round and smooth, the protruding processes are thick and long, the neuron activity is strong, and the phenomenon of apoptosis gradually occurs after the neurons cultured by resuscitation are cultured for 20 days.

Example 4

Example 4 differs from example 1 in that in the recovery step, the cryopreserved neurons were transferred to planting medium for culture. The mixture ratio of the planting culture medium, the neuron culture medium, the neural stem cell culture medium, the freezing medium and the resuscitation culture medium adopted in the embodiment 4 is as follows:

The frozen stock solution comprises the following components in percentage by volume: 55.00mL of neuronal medium, 45.00mL of 10% bovine serum albumin, and 5.50mL of dimethyl sulfoxide.

The neurons recovered in example 4 were subjected to immunofluorescence staining test (primary antibody: Tub-M1: 200GFAP-R1:200 secondary antibody: GM488GR 5681: 500), and the number of Tub-positive cells was calculated to be 90%, and the number of GFAP-positive cells was calculated to be 0%. The neuron cells cultured by resuscitation are round and smooth, the protruding processes are thick and long, the neuron activity is strong, and the phenomenon of apoptosis gradually occurs after the neurons cultured by resuscitation are cultured for 20 days.

Example 5

Example 5 differs from example 1 in that in the recovery step, the cryopreserved neurons were transferred to a planting medium for culture. The mixture ratio of the planting culture medium, the neuron culture medium, the neural stem cell culture medium, the freezing medium and the resuscitation culture medium adopted in the example 5 is as follows:

The frozen stock solution comprises the following components in percentage by volume: 45.00mL of neuronal medium, 49.50mL of 10% bovine serum albumin, 4.50mL of dimethyl sulfoxide.

The neurons recovered in example 5 were subjected to immunofluorescence staining test (primary antibody: Tub-M1: 200GFAP-R1:200 secondary antibody: GM488GR 5681: 500), and the number of Tub-positive cells and the number of GFAP-positive cells were measured to be 93% and 0%, respectively. The neuron cells cultured by resuscitation are round and smooth, the protruding processes are thick and long, the neuron activity is strong, and the phenomenon of apoptosis gradually occurs after the neurons cultured by resuscitation are cultured for 20 days.

Example 6

Example 6 differs from example 1 in that in the recovery step, the cryopreserved neurons were transferred to a planting medium for culture. The mixture ratio of the planting culture medium, the neuron culture medium, the neural stem cell culture medium, the freezing medium and the resuscitation culture medium adopted in the embodiment 6 is as follows:

The frozen stock solution comprises the following components in percentage by volume: 55.00mL of neuronal medium, 40.50mL of 10% bovine serum albumin, and 5.50mL of dimethyl sulfoxide.

The neurons recovered in example 6 were subjected to immunofluorescence staining test (primary antibody: Tub-M1: 200GFAP-R1:200 secondary antibody: GM488GR 5681: 500), and the number of Tub-positive cells was calculated to be 91% and the number of GFAP-positive cells was calculated to be 0%. The neuron cells cultured by resuscitation are round and smooth, the protruding processes are thick and long, the neuron activity is strong, and the phenomenon of apoptosis gradually occurs after the neurons cultured by resuscitation are cultured for 20 days.

Example 7

Example 7 differs from example 1 in that in the recovery step, the cryopreserved neurons were transferred to recovery medium for culture. The mixture ratio of the planting culture medium, the neuron culture medium, the neural stem cell culture medium, the freezing medium and the resuscitation culture medium adopted in the example 7 is as follows:

The volume ratio of the components of the resuscitation culture medium is as follows: 45.00mL of neuronal medium, 50.00mL of neural stem cell medium, 1.0mL of 10% BSA, and 1.0mL of 100 XNGF.

The neurons recovered in example 7 were subjected to immunofluorescence staining experiments (primary antibody: Tub-M1: 200GFAP-R1:200 secondary antibody: GM488GR 5681: 500), and by calculation, the total cell viability rate after cryopreservation reached 72%, the ratio of the number of Tub positive cells to the number of GFAP positive cells was 10:62, the recovered and cultured neuron cells were round and smooth, and the protruding processes were thick and long, which indicated that the neuron activity was strong, and a large number of astrocytes also survived, and thus the interaction between astrocytes and neurons was convenient to study. Experiments prove that the phenomenon of apoptosis gradually appears after the neurons and the astrocytes are recovered and cultured for 20 days, and the survival time of the neurons and the astrocytes in vitro is prolonged.

Example 8

Example 8 differs from example 1 in that in the recovery step, the cryopreserved neurons were transferred to recovery medium for culture. The mixture ratio of the planting culture medium, the neuron culture medium, the neural stem cell culture medium, the freezing medium and the resuscitation culture medium adopted in the embodiment 8 is as follows:

The volume ratio of the components of the resuscitation culture medium is as follows: 55.00mL of neuronal medium, 50.00mL of neural stem cell medium, 1.0mL of 10% BSA, and 1.0mL of 100 XNGF.

The neurons recovered in the example 8 are subjected to an immunofluorescence staining experiment (primary antibody: Tub-M1: 200GFAP-R1:200 secondary antibody: GM488GR 5681: 500), the total cell survival rate after cryopreservation reaches 77%, the ratio of the number of Tub positive cells to the number of GFAP positive cells is 12:65, the cells of the recovered and cultured neurons are round and smooth, and the protruding processes are thick and long, so that the neuron activity is strong, a large number of astrocytes can survive, and the interaction between the astrocytes and the neurons can be conveniently researched. Experiments prove that the phenomenon of apoptosis gradually appears after the neurons and the astrocytes are recovered and cultured for 20 days, and the survival time of the neurons and the astrocytes in vitro is prolonged.

Example 9

Example 9 differs from example 1 in that in the recovery step, the cryopreserved neurons were transferred to recovery medium for culture. The mixture ratio of the planting culture medium, the neuron culture medium, the neural stem cell culture medium, the freezing medium and the resuscitation culture medium adopted in the example 9 is as follows:

The neurons recovered in example 9 were subjected to immunofluorescence staining experiments (primary antibody: Tub-M1: 200GFAP-R1:200 secondary antibody: GM488GR 5681: 500), and by calculation, the total cell viability rate after cryopreservation reached 76%, the ratio of the number of Tub positive cells to the number of GFAP positive cells was 15:61, the recovered and cultured neuron cells were round and smooth, and the protruding processes were thick and long, which indicated that the neuron activity was strong, and a large number of astrocytes also survived, and thus the interaction between astrocytes and neurons was convenient to study. Experiments prove that the phenomenon of apoptosis gradually appears after the neurons and the astrocytes are recovered and cultured for 20 days, and the survival time of the neurons and the astrocytes in vitro is prolonged.

Comparative example 1

Comparative example 1 differs from example 1 in that in the recovery step, the neurons after cryopreservation were transferred to recovery medium for culture. The proportion of the planting culture medium, the neuron culture medium, the freezing medium and the resuscitation culture medium adopted in the comparative example 1 is as follows:

The volume ratio of the components of the resuscitation culture medium is as follows: 50.00mL of neuronal medium, 0.50mL 10% BSA, 0.5mL100 XNGF.

The neurons recovered in comparative example 1 were subjected to immunofluorescence staining assay (primary antibody: Tub-M1: 200GFAP-R1:200 secondary antibody: GM488GR 5681: 500), and the results are shown in FIG. 9, which indicates that the assay failed and no viable neurons were detected. Therefore, the recovery culture medium plays an important role in recovery of the neurons after cryopreservation, and not only are different types of cells recovered by different recovery culture media, but also survival of the neurons is influenced.

Comparative example 2

Example 2 differs from example 1 in that in the recovery step, one half of the wells were cultured with the plating medium for 4h followed by changing the neuron medium, and the other half of the wells were cultured with the plating medium with 0.1. mu.L of 10000 XRA, 10. mu.L of 100 XNGF added for 4h followed by changing the neuron medium. The proportion of the planting culture medium, the neuron culture medium and the freezing medium adopted in the comparative example 2 is as follows:

The frozen stock solution comprises the following components in percentage by volume: 45.00mL 10% bovine serum albumin, 5.00mL dimethyl sulfoxide.

The neurons recovered in comparative example 2 were subjected to immunofluorescence staining assay (primary antibody: Tub-M1: 200GFAP-R1:200 secondary antibody: GM488GR 5681: 500), and the results are shown in FIG. 10, indicating that the assay failed and no viable neurons were detected. Compared with example 1, in comparative example 2, because of different freezing medium adopted, the cells are damaged and even die when the neurons are frozen, and the neurons can not survive in the resuscitation culture.

Comparative example 3

Comparative example 3 differs from example 1 in that in the resuscitation step, one half of the wells were incubated with resuscitation medium and the other half of the wells were incubated with resuscitation medium supplemented with 0.1. mu.L of 10000 × RA, 10. mu.L of 100 × NGF. The proportion of the planting culture medium, the neuron culture medium, the neural stem cell culture medium, the freezing medium and the resuscitation culture medium adopted in the comparative example 3 is as follows:

The volume ratio of the recovery culture medium is as follows: 45.00mL of neuronal medium, 5.00mL of neural stem cell medium, 2.5mL of 10% BSA.

The neurons recovered in comparative example 3 were subjected to immunofluorescence staining assay (primary antibody: Tub-M1: 200GFAP-R1:200 secondary antibody: GM488GR 5681: 500), and the results of the assay showed that the assay failed and no viable neurons were detected as shown in FIG. 11.

In contrast to example 1, comparative example 3, which uses a frozen stock solution, failed to resuscitate neurons even with a modified resuscitation medium.

Comparative example 4

Comparative example 4 differs from example 1 in that in the recovery step, one half of the wells were cultured with the plating medium for 4h and then with the neuron medium, and the other half of the wells were cultured with the plating medium with 0.1. mu.L of 10000 XRA and 10. mu.L of 100 XNGF added for 4h and then with the neuron medium. The proportion of the planting culture medium, the neuron culture medium and the freezing medium adopted in the comparative example 4 is as follows:

The frozen stock solution comprises the following components in percentage by volume: 45.00mL of neuronal medium, 5.00mL of dimethyl sulfoxide.

The neurons recovered in comparative example 4 were subjected to immunofluorescence staining experiments (primary antibody: Tub-M1: 200GFAP-R1:200 secondary antibody: GM488GR 5681: 500), indicating that the experiments failed and no viable neurons were detected.

Comparative example 5

Comparative example 5 example 1 differs in that in the recovery step, the neurons after cryopreservation were transferred to recovery medium for culture. The proportion of the planting culture medium, the neuron culture medium, the freezing medium and the resuscitation culture medium adopted in the comparative example 5 is as follows:

The frozen stock solution comprises the following components in percentage by volume: 450mL of neuronal medium, 5.00mL of dimethyl sulfoxide.

The neurons threatened in comparative example 5 were subjected to an immunofluorescence staining experiment (primary antibody: Tub-M1: 200GFAP-R1:200 secondary antibody: GM488GR 5681: 500), indicating that the experiment failed and no viable neurons were detected. Therefore, the recovery culture medium plays an important role in recovery of the neurons after cryopreservation, and not only are different types of cells recovered by different recovery culture media, but also survival of the neurons is influenced.

The survival rate of the cryopreserved neurons can be obtained by further calculating the ratio of the number of Tub positive cells obtained by resuscitating and culturing primary neurons after being frozen for a certain time to the number of Tub positive cells obtained by directly planting and culturing the primary neurons, and the survival rates of the neurons in the frozen storage period of 1 month, 6 months, 12 months and 24 months are respectively determined in examples 1 and 2, and the results are shown in Table 1.

TABLE 1 example 1 cell viability at different cryopreservation periods

As can be seen from Table 1, the cryopreservation solution of the invention can keep neurons alive for a long time, and is convenient for experimental research and sample preservation.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

Claims

1. The neuron cryopreservation liquid is characterized by comprising the following components in parts by volume: 45.00-55.00 parts of neuron culture medium, 40.50-49.50 parts of 10% bovine serum albumin and 4.50-5.50 parts of dimethyl sulfoxide; the neuron cryopreservation liquid does not contain glutamine; the neuron culture medium comprises the following components in parts by volume: 45.00-55.00 parts of Neurobasal A culture medium or Neurobasal culture medium and 0.90-1.10 parts of B27 cell culture additive.

2. The neuron cryopreservation solution of claim 1, wherein the neuron culture medium further comprises the following components in parts by volume: 0.27 to 0.33 portions of double antibody.

3. The neuronal cryopreservation liquid according to claim 1 or 2, wherein the Neurobasal A medium is glycoform.

4. A freezing and thawing method for neurons is characterized by comprising the following steps:

freezing and storing: mixing the primary neurons with the cryopreservation solution of any one of claims 1 to 3 to prepare a cryopreserved cell sap, placing the cryopreserved cell sap in a cryopreservation tube, freezing the cryopreserved cell sap in a refrigerator at the temperature of-20 ℃ for 1.0 to 2.0 hours, and freezing the cryopreserved cell sap in a refrigerator at the temperature of-80 ℃;

5. The neuron cryopreservation and resuscitation method according to claim 4, further comprising, before the cryopreservation step, the steps of: and mixing the primary neurons with the planting culture medium, centrifuging at 800-1200 rpm for 4-6 min, and removing a supernatant after centrifuging.

6. The neuron cryopreservation and recovery method according to claim 4, wherein before the neuron after cryopreservation is transferred to a recovery medium culture or a planting medium culture, the recovery method further comprises the following steps: mixing the frozen neurons with a resuscitation medium, centrifuging at 800-1200 rpm for 4-6 min, and removing a supernatant after centrifugation.

7. The method of freezing and resuscitating neurons of claim 4, wherein the neural tissue comprises any one of hippocampal tissue, cerebral cortex, and brain tissue.

8. The neuron cryopreservation and recovery method according to claim 4, wherein the planting medium comprises the following components in parts by volume: 36.53-45.87 parts of DMEM medium, 4.50-5.50 parts of horse serum and 2.70-3.30 parts of fetal bovine serum.

9. The method for freezing and thawing the neurons according to any of claims 4 to 8, wherein in the thawing step, the frozen neurons are transferred to a planting medium for culture for 4 hours and then are replaced by a neuron medium for culture.