CN115251044A - Cell vitrification preservation method based on hydrogel film encapsulation - Google Patents
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/0231—Chemically defined matrices, e.g. alginate gels, for immobilising, holding or storing cells, tissue or organs for preservation purposes; Chemically altering or fixing cells, tissue or organs, e.g. by cross-linking, for preservation purposes
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0278—Physical preservation processes
- A01N1/0284—Temperature processes, i.e. using a designated change in temperature over time
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Abstract
The invention relates to a cell vitrification preservation method based on hydrogel film encapsulation, belonging to the field of low-temperature biomedical engineering. The specific operation steps are as follows: (1) Uniformly mixing a chitosan solution and a hydroxyethyl cellulose solution to obtain a matrix solution; (2) Adding cells to be preserved into the matrix solution, dropwise adding a beta sodium glycerophosphate solution, and obtaining a suspension solution under an ice bath condition; (3) Transferring the suspension solution into a film-forming mold, standing at a constant temperature of 37 ℃ in an aseptic environment until a hydrogel film is formed, and performing film-forming encapsulation on the cells to be stored in the hydrogel film; (4) Transferring to a freezing slide glass, putting the freezing slide glass with the hydrogel film into liquid nitrogen, and rapidly cooling to below-150 ℃ for glass transition; and then transferring the mixture into a deep low temperature refrigerator or a liquid nitrogen tank to realize long-term storage. When the temperature needs to be restored, taking out the mixture, quickly restoring the temperature in a constant-temperature water bath kettle at 37-40 ℃ and restoring the temperature to be liquid; the cell survival rate before and after vitrification preservation has no significant difference.
Description
Technical Field
The invention belongs to the field of low-temperature biomedical engineering, and particularly relates to a high-flux protective agent-free vitrification preservation method based on hydrogel thin-film encapsulation, which is used for preserving cells at low temperature.
Background
At present, the traditional slow cooling method is generally adopted in the low-temperature preservation industry, low-temperature damage caused by factors such as ice crystal growth is difficult to avoid, the effect is poor in preservation of many types of cells, and the cooling process is long and low in efficiency. The vitrification preservation is a revolutionary method, avoids the growth of ice crystals by promoting the vitrification transformation of preserved objects, has principle advantages compared with the traditional method, and is an important direction for the development of the low-temperature preservation technology.
The high-speed temperature reduction and rewarming are the key for realizing vitrification preservation, and the efficiency of two heat transfer processes of convective heat transfer between the temperature reduction rewarming working medium and a preserved object carrier and heat transfer inside the preserved object is the core of influencing the temperature reduction rewarming speed. At present, the method adopts the trace technology (such as a microtube method and a micro-droplet method) of the preserved object in the industry, and the temperature reduction and rewarming rate is improved by reducing the size (generally below microliter) of the preserved object and reducing the internal thermal conduction resistance. Although the micro-quantification technology successfully realizes the vitrification preservation of some preserved objects, the micro-quantification technology has significant application limitations, particularly has severe limitation (less than 1 microliter) on the volume of the preserved objects, and is generally only suitable for preserving special samples (such as 1 or a plurality of embryonic stem cells).
On the other hand, the prior vitrification preservation method generally uses a protective agent with higher concentration to inhibit the growth of ice crystals and further promote vitrification effect. However, the high-concentration protective agent not only has serious toxic and side effects, but also increases the difficulty of adding and removing the protective agent, so that the quality and the effect of vitrification preservation are not ideal.
Disclosure of Invention
The invention provides a cell vitrification preservation method based on hydrogel film encapsulation, aiming at solving the problems that the existing vitrification preservation treatment efficiency is poor, the flux is low, and the serious influence of toxic and side effects of a protective agent is caused.
A cell vitrification preservation method based on hydrogel film encapsulation is characterized by comprising the following operation steps:
(1) Preparation of matrix solution
Dissolving chitosan in dilute acetic acid to obtain a chitosan solution with the mass concentration of 2%; dissolving hydroxyethyl cellulose by using distilled water to obtain a hydroxyethyl cellulose solution with the concentration of 10 mg/mL-20 mg/mL, and uniformly mixing 6mL of chitosan solution and 1mL of hydroxyethyl cellulose solution to obtain 7mL of matrix solution;
(2) Preparation of suspension solution
Washing and resuspending cells to be preserved by using normal saline, adding 1mL of cells to be preserved into 7mL of matrix solution, dropwise adding 1mL of beta sodium glycerophosphate solution with the mass concentration of 40-56%, and uniformly stirring under an ice bath condition to obtain a suspension solution;
(3) Hydrogel film preparation and thin film encapsulation
Transferring the suspension solution into a thin film mold, standing at a constant temperature of 37 ℃ in an aseptic environment, and carrying out hydrogen bonding reaction on chitosan and beta sodium glycerophosphate until a hydrogel film is formed, so that cells to be stored are packaged in the hydrogel film in a thin film manner; the cell survival rate before and after the thin-film encapsulation has no obvious difference;
(4) Rapid cooling and vitrification
Transferring the hydrogel film to a freezing slide glass, putting the freezing slide glass with the hydrogel film into liquid nitrogen, and rapidly cooling to below-150 ℃ to realize glass transition; then transferring the vitrified hydrogel film into a deep low temperature refrigerator or a liquid nitrogen tank along with the frozen slide glass for long-term storage;
when the temperature is required to be restored, taking out the stored hydrogel film, quickly restoring the temperature in a constant-temperature water bath kettle at 37-40 ℃, and then restoring the hydrogel film to be liquid by cooling or shaking and other modes; the cell survival rate before and after vitrification preservation has no significant difference.
The further technical scheme is as follows:
in the step (1), in the preparation of the matrix solution, chitosan with deacetylation degree of 85-95% and Mn =50000 is added into dilute acetic acid with concentration of 0.1M to obtain a 2% chitosan solution with mass fraction.
In the step (3), the film-forming die is a disc-shaped flat-bottom shallow slot, the depth of the flat-bottom shallow slot is less than 1mm, and the width and the length of the flat-bottom shallow slot are more than 1cm; the thickness of the formed hydrogel film is less than 1mm.
In the step (4), the frozen slide glass is made of a common glass slide, a quartz slide or a high-temperature ceramic sheet, and the thickness of the frozen slide glass is less than 0.2mm.
And (4) synchronously cooling the plurality of frozen slides, and rapidly cooling the plurality of frozen slides in a micro-channel, micro-jet array and micro-spray enhanced heat exchange mode.
And (4) synchronously carrying out rewarming on a plurality of frozen slides, and carrying out rewarming more quickly by a micro-channel, a micro-jet array and a micro-spray enhanced heat exchange mode.
The beneficial technical effects of the invention are embodied in the following aspects:
1. the invention uses temperature sensitive hydrogel material to package cells for vitrification preservation, the material generates sol-gel conversion above critical temperature to form hydrogel, has 3D network structure, has stronger constraint effect on water molecules, can inhibit the generation of ice crystals inside and outside cells in cooling, promotes vitrification transformation, can inhibit reverse vitrification in the process of rewarming, and has good biocompatibility, thereby improving the survival rate of cells in the preservation process, avoiding the toxic damage brought by the traditional protective agent, and avoiding the problem of removing the protective agent.
2. Through a thin-film packaging form, the effective heat exchange area of the surface is increased while the internal heat conduction path of the sample is reduced, and meanwhile, the internal heat conduction thermal resistance and the surface heat exchange thermal resistance in the cooling and rewarming processes obviously improve the heat transfer efficiency in vitrification storage and are beneficial to realizing the rapid cooling and rapid rewarming required by the vitrification transformation of the sample; meanwhile, the length and the width of the packaging film can be flexibly extended to realize the treatment of the preserved object with larger volume, the limitation of the micro-tube method or the micro-droplet method on the volume of the preserved object is broken through, and the vitrification preservation treatment flux is remarkably improved.
Drawings
FIG. 1 is a graph of concentration and viability of fresh cells under bright field and fluorescence.
FIG. 2 is a drawing of a hydrogel film mold.
FIG. 3 is a diagram of hydrogel encapsulated cells under a microscope.
Fig. 4 is a frozen slide image.
FIG. 5 is a diagram of a hydrogel film clamped using two frozen slides.
FIG. 6 is a graph showing the concentration and viability of hydrogel films in saline at the light field and fluorescence of cells after rewarming.
Detailed description of the invention
The invention is further described below by way of examples.
Example 1
Description of the materials and operating procedures used in example 1:
the mesenchymal stem cells are from Shanghai Jinning industry Co., ltd, DMEM, fetal calf serum, cryoprotectant, pancreatin, sodium alginate, chitosan, AO-PI reagent, EDTA, sodium citrate, calcium chloride, beta-sodium glycerophosphate, hydroxyethyl cellulose and normal saline. Those without the condition indication can be performed according to the conventional conditions or the conditions recommended by the manufacturer. The reagents used are not indicated by manufacturers, and conventional products sold in the market can be purchased.
The operation steps of the vitrification preservation operation of the cells based on the hydrogel film encapsulation are as follows:
(1) Preparation of matrix solution
Weighing 2g of chitosan with deacetylation degree of 90% and Mn =50000, adding into 100ml of 0.1M acetic acid solution for dissolving, stirring for 2h by using a magnetic particle, filtering and sterilizing at 4 ℃, and preparing into a chitosan solution with mass concentration of 2%; weighing 10mg of hydroxyethyl cellulose (HEC), sterilizing and dissolving in distilled water to obtain a hydroxyethyl cellulose solution with the concentration of 10 mg/ml; and fully stirring and uniformly mixing 6mL of chitosan solution and 1mL of hydroxyethyl cellulose solution to obtain a matrix solution.
(2) Preparation of the suspension solution
Inoculating stem cells to be stored into a complete cell culture medium, wherein the inoculation amount is 2.5 x 10^6, the complete cell culture medium is prepared by uniformly mixing a DMEM culture medium with the volume of 90% and a fetal bovine serum culture medium with the volume of 10%, culturing for 3d, and observing the growth condition; when the cells grow to 70% -90% of fusion, collecting fresh expanded stem cells, removing the cell culture medium, and washing off the redundant cell culture medium by using physiological saline; adding 2ml of pancreatin with the mass concentration of 0.25% to digest and amplify the stem cells, rounding the amplified stem cells, and stopping the digestion by using a cell complete culture medium; collecting the digested stem cells, centrifuging at 1000r/min for 5min, and removing supernatant; resuspending and centrifuging once with physiological saline, removing the supernatant, resuspending the cells again with 5ml of physiological saline to obtain resuspended cells, and staining the resuspended cells with a live-dead-cell fluorescent dye (APExBIO), as shown in FIG. 1, wherein a in FIG. 1 is the state of the resuspended cells in the bright field, and b in FIG. 1 is the state of the resuspended cells under fluorescence after staining. Detecting the survival rate and concentration of the resuspended stem cells by using a cell counting instrument, wherein the concentration of the resuspended stem cells is 2.43 x 10^ 6/ml, and the survival rate is 98.7 percent;
adding 1mL of the resuspended stem cells into 7mL of the matrix solution, uniformly mixing, and standing for 15min to obtain a mixed solution; detecting the cell viability rate of the mixed solution, wherein the cell viability rate is 97.30%; dropwise adding 1mL of sterilized beta-sodium glycerophosphate solution with the mass fraction of 56% into the obtained mixed solution, and stirring for 20min in ice bath to obtain a suspension solution; sampling is carried out, and the survival rate of the cells in the suspension solution is measured to be 97.10%.
(3) Hydrogel film preparation and thin film encapsulation
Referring to fig. 2, the prepared suspension solution was transferred to a hydrogel film mold, referring to fig. 4, which was a disc-shaped shallow flat bottom groove having a depth of less than 1mm, a width of 24mm, and a length of 60mm. Placing the hydrogel film in a constant-temperature sterile environment at 37 ℃, and observing the formation of the hydrogel film; the thickness of the hydrogel film is less than 1mm. Referring to fig. 3, under a microscope, the stem cells to be preserved are encapsulated in the hydrogel film in a thin film manner, and sampling and detecting are carried out, wherein the stem cell survival rate is 98.90%.
(4) Rapid cooling and vitrification
Referring to fig. 5, the hydrogel film was transferred to a freezing slide, the freezing slide with the hydrogel film was clamped, and put into liquid nitrogen to rapidly cool to-196 ℃, thereby achieving vitrification.
The material of freezing slide glass material, thickness is 0.15mm.
Transferring the vitrified hydrogel film along with the frozen slide glass into a deep low temperature refrigerator or a liquid nitrogen tank for long-term storage.
When the rewarming is needed, the vitrified hydrogel film is taken out along with the frozen slide, put into the physiological saline with the temperature of 37 ℃ prepared in advance for quick rewarming, and sampled to detect the survival rate, wherein the survival rate is 97.60 percent, as shown in fig. 6, a in fig. 6 is the state of rewarming cells in a bright field, and b in fig. 1 is the state of rewarming cells under fluorescence after staining. After being placed for 1h at normal temperature, the detection activity rate is 94.87%; the mixture is continuously placed for 1h and is detected again, and the activity rate is 93.67 percent. The method can directly infuse human body without cleaning cells after cell resuscitation, and can avoid the problem of removing protective agent and mechanical injury caused by centrifugation after rewarming.
When the thickness of the film is kept to be less than 0.2mm, the hydrogel film packaged cells with different volumes can be ensured to have smaller internal heat conduction thermal resistance and surface heat exchange heat and negative under different sample volume conditions and maintain the same high-efficiency heat transfer efficiency.
Example 2
As a control, stem cells were subjected to a temperature-decreasing and rewarming treatment in the same manner using a commercially available cryoprotectant as a control:
(1) Collecting stem cells: the viability rate of the cells was found to be 94.67% when fresh cells were collected, and after removal of the supernatant, the cells were suspended by adding cryoprotectant at the usual storage concentration of 1 x 10^ 7/mL, at which time the viability rate was 92.17%. Transferring the cells to a freezing tube, and marking;
(2) Cooling: putting the collected stem cells into liquid nitrogen at the temperature of-196 ℃ for rapid cooling and low-temperature preservation;
(3) Rewarming: rapidly rewarming the cryopreserved stem cells in a water bath kettle at 37 ℃, and detecting the survival rate, wherein the survival rate is 89.80%; after the stem cells subjected to rewarming are placed for 1 hour at normal temperature, the survival rate is 81.50 percent; after being placed for 1 hour at normal temperature, the cell survival rate is 74.37 percent
Therefore, on the premise of the same cooling and rewarming operation, the hydrogel thin-film encapsulation preservation effect is better, and the cell survival rate after hydrogel encapsulation preservation under the same placing condition (temperature and time) is obviously higher than that after the cell survival rate after the cell preservation by the common cryoprotectant.
It will be readily understood by those skilled in the art that the above embodiment 1 is merely a preferred embodiment of the present invention, and is not intended to limit the present invention, and any modifications, equivalents, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (6)
1. A cell vitrification preservation method based on hydrogel film encapsulation is characterized by comprising the following operation steps:
(1) Preparation of matrix solution
Dissolving chitosan in dilute acetic acid to obtain a chitosan solution with the mass concentration of 2%; dissolving hydroxyethyl cellulose by using distilled water to obtain a hydroxyethyl cellulose solution with the concentration of 10 mg/mL-20 mg/mL, and uniformly mixing 6mL of chitosan solution and 1mL of hydroxyethyl cellulose solution to obtain 7mL of matrix solution;
preparation of suspension solution
Washing and resuspending cells to be preserved by using normal saline, adding 1mL of cells to be preserved into 7mL of matrix solution, dropwise adding 1mL of beta sodium glycerophosphate solution with the mass concentration of 40-56%, and uniformly stirring under an ice bath condition to obtain a suspension solution;
hydrogel film preparation and thin film encapsulation
Transferring the suspension solution into a thin film mold, standing at a constant temperature of 37 ℃ in an aseptic environment, and carrying out hydrogen bonding reaction on chitosan and beta sodium glycerophosphate until a hydrogel film is formed, so that cells to be stored are packaged in the hydrogel film in a thin film manner; the cell survival rate before and after the thin-film encapsulation has no obvious difference;
rapid cooling and vitrification
Transferring the hydrogel film to a freezing slide glass, putting the freezing slide glass with the hydrogel film into liquid nitrogen, and rapidly cooling to below-150 ℃ to realize glass transition; then transferring the vitrified hydrogel film into a deep low-temperature refrigerator or a liquid nitrogen tank along with the frozen slide glass to realize long-term storage;
when the temperature needs to be restored, taking out the stored hydrogel film, quickly restoring the temperature in a constant-temperature water bath kettle at 37-40 ℃, and then restoring the hydrogel film to be liquid by cooling or shaking and other modes; there was no significant difference in cell viability before and after vitrification preservation.
2. The method for preserving cells by vitrification based on the encapsulation of hydrogel film according to claim 1, characterized in that: in the step (1), in the preparation of the matrix solution, chitosan with deacetylation degree of 85-95% and Mn =50000 is added into dilute acetic acid with concentration of 0.1M to obtain a chitosan solution with mass fraction of 2%.
3. The method for preserving cells by vitrification based on the encapsulation of hydrogel film according to claim 1, characterized in that: in the step (3), the film-forming die is a disc-shaped flat-bottom shallow slot, the depth of the flat-bottom shallow slot is less than 1mm, and the width and the length of the flat-bottom shallow slot are more than 1cm; the thickness of the formed hydrogel film is less than 1mm.
4. The method for preserving cells by vitrification based on the encapsulation of hydrogel film according to claim 1, characterized in that: in the step (4), the frozen slide glass is made of a common glass slide, a quartz slide or a high-temperature ceramic sheet, and the thickness of the frozen slide glass is less than 0.2mm.
5. The method for preserving cells by vitrification based on the encapsulation of hydrogel film according to claim 1, characterized in that: and (4) synchronously cooling the plurality of frozen slides, and rapidly cooling the plurality of frozen slides in a micro-channel, micro-jet array and micro-spray enhanced heat exchange mode.
6. The method for preserving cells by vitrification based on the encapsulation of hydrogel film according to claim 1, characterized in that: and (4) synchronously carrying out rewarming on a plurality of frozen slides, and carrying out rewarming more quickly by a micro-channel, a micro-jet array and a micro-spray enhanced heat exchange mode.
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