CN113234658B

CN113234658B - Grinding-based method for preparing viable single cells

Info

Publication number: CN113234658B
Application number: CN202110735877.5A
Authority: CN
Inventors: 赵伟; 余嘉莹; 廖东升; 邱坤
Original assignee: Chengdu Daosheng Biotechnology Co ltd
Current assignee: Chengdu Daosheng Biotechnology Co ltd
Priority date: 2021-06-30
Filing date: 2021-06-30
Publication date: 2023-04-07
Anticipated expiration: 2041-06-30
Also published as: CN113234658A

Abstract

The application discloses a grinding-based method for preparing viable single cells, which specifically comprises the following operation steps: preparing grinding materials, including sterilized living biological tissue blocks, cutting the biological tissue blocks into a plurality of small blocks with the length not more than 2mm by using sterile ophthalmic scissors, cleaning, putting the small blocks into a loosening reagent, soaking for 20-30min, then putting the small blocks into a centrifugal tube, centrifuging, putting the centrifugal tube into a grinding device, grinding, filtering the obtained tissue blocks, cell clusters and single cell mixture by using a cell screen, collecting filtrate after filtration, centrifuging again, collecting single cells, and storing for later use. The grinding device provided by the invention can realize overturning grinding of biological tissues by matching with the unique shear magnetic beads, so that the biological tissues have the advantages of high single cell rate and high survival rate after being ground.

Description

Grinding-based method for preparing viable single cells

Technical Field

The invention relates to the technical field of biology, in particular to the technical field of single cell preparation, and specifically relates to a grinding-based method for preparing viable single cells.

Background

Biological tissue processing is one of the most routine laboratory experiments, and many biological experiments need to be performed on a cell suspension basis. With the development of technology, according to the different laboratory specifications and requirements, the technology of biological tissue treatment, cell disruption and single cell treatment has become correspondingly abundant. The common physical treatment methods comprise three methods of grinding, net rubbing and shaking, and the more advanced method adopts an ablation technology to carry out tissue unicellularization, but the conventional physical enzyme method still occupies most laboratory users.

The existing laboratory grinding is still in a manual operation state, in order to improve the yield of unicellularization, an enzyme method and physical grinding are usually adopted for cooperative operation, and as the quality of a unicellular suspension almost directly influences subsequent biological experiments, how to obtain higher unicellular rate and even keep as many viable unicells as possible is a technical problem to be solved in the existing single cell suspension preparation field, and meanwhile, the technical bottleneck is also based on the subsequent unicellular culture.

Disclosure of Invention

In order to solve the problems of low yield and instability of the survival single cell obtained by preparing the single cell suspension in the prior art, the application provides a grinding-based method for preparing the survival single cell, which is used for replacing the traditional artificial grinding to prepare the single cell suspension, and the survival single cell is obtained after conventional filtration and centrifugation treatment and is used for subsequent biological experiments. The conventional single cell acquisition method almost comprises the steps of preparing a single cell suspension in advance, and then obtaining the single cells with higher concentration in the obtained cell suspension in conventional manners such as centrifugation, and the like, and of course, the conventional method for obtaining the single cells based on ultrasonic ablation also successively appears in the prior art, but the ultrasonic ablation instrument and equipment are high in price and are not suitable for general purchase in conventional laboratories; therefore, the conventional mechanical method, the wire-twisting method and the grinding method are mostly adopted. The preparation method of the survival single cell provided by the invention aims to replace the traditional mechanical method, the net rubbing method and the grinding method, and solves the problems of unstable yield, complex operation flow, serious influence of experience and expertise of operators on the quality of the obtained suspension and the like caused by the traditional manual operation.

In order to achieve the purpose, the technical scheme adopted by the application is as follows:

a method for preparing survival single cells based on grinding specifically comprises the following operation steps:

step ST100, preparing grinding materials including sterilized living biological tissue blocks, a loosening agent and a grinding device; the debonding reagent consists of neutral proteinase in the final concentration of 2mg/mL, type I collagenase in the final concentration of 0.09mg/mL, sodium chloride in the final concentration of 6-10mg/mL and magnesium chloride in the final concentration of 2-5 mM/L.

ST200, cutting the sterilized and surviving biological tissue blocks into a plurality of small blocks with the length not more than 2mm by using sterile ophthalmic scissors, then cleaning the small blocks by using normal saline, and soaking the cleaned biological tissue blocks in a loosening reagent for 20-30min, wherein the temperature environment during the soaking is 18-24 ℃;

step ST300, placing the soaked biological tissue block and the loosening reagent in a centrifugal tube, and adding a plurality of shearing magnetic beads into the centrifugal tube; the shear magnetic beads comprise glass beads with equal diameters and PP beads which are mixed according to the proportion of 0.5-0.8 ³ The spheroid of (4); meanwhile, the shear magnetic beads may further include spherical zirconia having an equal diameter for different biological tissues.

ST400, placing the centrifugal tube into a grinding device for grinding, wherein the grinding device at least has back and forth oscillation of more than two moving degrees of freedom simultaneously or alternatively applied to the centrifugal tube, and the grinding time is 20-40min;

and step ST500, filtering the tissue block, the cell mass and the single cell mixture obtained in the step ST400 by using a cell screen, collecting the filtered filtrate, centrifuging, collecting the single cell, and storing for later use.

In the preparation method, a great deal of experimental research is carried out in step ST300 to obtain the rule of the shear magnetic beads on the fragmentation and dispersion of the biological tissue, so that the shear magnetic beads are improved. The research of the applicant finds that the mode of adding the shear magnetic beads and matching with the oscillation can be applied to the conventional cell disruption, relatively ideal living single cells of biological tissues can be obtained by improving the magnetic beads and the grinding mode, and the obtained cells can keep the living state for a long time. The magnetic bead grinding method is mainly characterized in that irregular oscillation of magnetic beads is utilized, biological tissues are cut through collision to achieve the purpose of crushing, the magnetic bead grinding method is often used for extracting DNA and RNA in organelles or cells, the cells can be quickly damaged under the action of the magnetic beads, and the purpose of directional extraction can be achieved by adding related biological agents or enzymes, so that the magnetic bead crushing mode is widely applied to the field of cell crushing and DNA and RNA extraction. However, in order to bring obvious damage to cells in such a way, grinding extraction by magnetic beads is hardly found in the field of living single cells; the applicant finds that single cells which can survive for a long time exist in single cell suspension prepared by grinding biological tissue blocks, and on the basis, in order to realize the automatic preparation of the single cells, the applicant can obtain the technical effect that the single cell rate is obviously higher than that of manual grinding by matching the developed grinding device with the improvement of shearing magnetic beads to grind the biological tissue. More importantly, the grinding process adopted by the invention can realize overturning grinding, namely the biological tissue can realize horizontal and up-and-down irregular overturning movement under the combined action of vibration and shearing magnetic beads in the grinding process, and can realize very uniform discrete action on the biological tissue under the frequent action of shearing magnetic beads. While the effect of ordinary grinding on biological tissues is relatively non-uniform with cells as a unit, there are a large number of cell clusters that cannot be ground efficiently. Therefore, the grinding mode has a very important role in obtaining more and more complete single cells, and for this reason, the applicant designs a brand-new grinding device aiming at the requirement. The method comprises the following specific steps:

the grinding apparatus in step ST100 includes a first oscillating mechanism providing a horizontal reciprocating motion and a second oscillating mechanism providing a vertical reciprocating motion. The first vibrating mechanism plays a role in shearing biological tissues by utilizing the inertia of shearing magnetic beads, and the second vibrating mechanism plays a role in overturning the biological tissues, so that the shearing magnetic beads have more uniform and comprehensive effects on the biological tissues, shearing blind areas are eliminated, and more single cells can be obtained. In order to realize above-mentioned technological effect, the grinder that this application adopted includes first driver, the pivot of first driver has linked firmly the driving-disc, the driving-disc supports to lean on first vibration mechanism through the level and smooth undulant lateral wall that has and is reciprocating motion for the driving-disc, first vibration mechanism slidable mounting is on the carousel that uses the driving-disc to install as the center, the carousel lower surface is connected with the casing, the casing with fixed connection can be dismantled to first driver.

Preferably, first shake mechanism is in including sliding the T shape guide rail in the spout, the T shape guide rail is close to the one end of driving-disc is provided with first fixed pulley, and the other end fixedly connected with of T shape guide rail is used for installing the centrifuge tube rack of centrifuging tube.

It is further preferred, first shock mechanism still includes canceling release mechanical system, canceling release mechanical system includes fixed connection and is in the first fixing base of carousel lower surface, fixed connection be in second fixing base on the T shape guide rail, anti-roll bar has been linked firmly on the second fixing base, first fixing base on have with the blind hole of anti-roll bar coaxial arrangement, the cover is equipped with the spring on the anti-roll bar, the both ends of spring respectively with blind hole bottom and second fixing base fixed connection on the first fixing base, the free end of anti-roll bar extends and inserts to the blind hole in to first fixing base direction.

Realize the motion of overturning of biological tissue piece in grinding process for better realization, preferably, still include the second and vibrate the mechanism, the second vibrates the mechanism including setting up the pulley support of carousel lower surface is provided with the second fixed pulley on the pulley support, the second fixed pulley through with the level and smooth undulation curved surface contact promotion that T shape guide rail lower surface set up vibrations about the T shape guide rail, there is the clearance between the upper surface of guide rail and the spout inner wall on the carousel, install in the clearance and promote all the time the reed is hugged closely with the carousel to the track downwards.

Has the advantages that:

according to the invention, the grinding device is matched with the shearing magnetic beads to realize uniform dispersion on biological tissues, and obtain the undamaged and alive single cells, compared with the existing grinding or magnetic bead grinding technology, the method saves the complexity of manual grinding, and eliminates the limitation that the existing magnetic bead grinding is only suitable for cell crushing and extraction of substances in cells.

The grinding device provided by the invention can realize overturning grinding of biological tissues by matching with the unique shear magnetic beads, so that the biological tissues have the advantages of high single cell rate and high survival rate after being ground.

The preparation method of the survival single cell provided by the invention does not need manual operation and is not influenced by manual operation techniques and experiences, and the occupancy ratio of the survival single cell in the ground cell suspension is high.

Drawings

In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings needed to be used in the description of the embodiments or the prior art will be briefly introduced below, it is obvious that the drawings in the following description are only some embodiments of the present application, and for those skilled in the art, other drawings can be obtained according to the drawings without inventive exercise.

FIG. 1 is a perspective isometric view of the present invention;

FIG. 2 is a top view of FIG. 1;

FIG. 3 is another visual isometric view of FIG. 1 (without the housing);

FIG. 4 is an enlarged view of the structure of region A in FIG. 3;

FIG. 5 is an exploded view of FIG. 3;

FIG. 6 is an exploded view of a first oscillating mechanism;

FIG. 7 is a microscopic enlarged view of the cell suspension obtained in the twined group in example 4;

FIG. 8 is a microscopic image of the cell suspension obtained from the ground group in example 4;

FIG. 9 is an enlarged view of a cell suspension obtained by the present invention under a microscope.

In the figure: 1-a shell; 2-a first oscillating mechanism; 21-T-shaped guide rails; 211-smooth relief surfaces; 22-a first fixed pulley; 23-centrifuge tube rack; 24-centrifuging the tube; 3, rotating a disc; 4-a pressure spring piece; 5-a first driver; 6-a resetting mechanism; 61-a first fixed seat; 62-a spring; 63-anti-roll bar; 64-a second fixed seat; 7-a second oscillating mechanism; 71-a pulley support; 72-a second fixed pulley; 8-drive plate.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are some embodiments of the present application, but not all embodiments. The components of the embodiments of the present application, generally described and illustrated in the figures herein, can be arranged and designed in a wide variety of different configurations.

Thus, the following detailed description of the embodiments of the present application, presented in the accompanying drawings, is not intended to limit the scope of the claimed application, but is merely representative of selected embodiments of the application. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.

It should be noted that: like reference numbers and letters refer to like items in the following figures, and thus, once an item is defined in one figure, it need not be further defined and explained in subsequent figures.

In the description of the present application, it should be noted that if the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc. indicate an orientation or positional relationship based on that shown in the drawings or that the product of the application is used as it is, this is only for convenience of description and simplicity of description, and does not indicate or imply that the device or element referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore should not be construed as limiting the present application. Furthermore, the appearances of the terms "first," "second," and the like in the description herein are only used for distinguishing between similar elements and are not intended to be construed as indicating or implying relative importance.

Furthermore, the terms "horizontal", "vertical" and the like when used in the description of the present application do not require that the components be absolutely horizontal or overhanging, but may be slightly inclined. For example, "horizontal" merely means that the direction is more horizontal than "vertical" and does not mean that the structure must be perfectly horizontal, but may be slightly inclined.

In the description of the present application, it should also be noted that, unless otherwise explicitly stated or limited, the terms "disposed," "mounted," "connected," and "connected" should be interpreted broadly, e.g., as being fixedly connected, detachably connected, or integrally connected; can be mechanically or electrically connected; they may be connected directly or indirectly through intervening media, or they may be interconnected between two elements. The specific meaning of the above terms in the present application can be understood in a specific case by those of ordinary skill in the art.

In order to fully illustrate that the method for preparing viable single cells by grinding provided by the invention can obtain a single cell yield and a survival rate which are obviously higher than those of the conventional manual grinding or net rubbing method, the following practical examples are used for one by one.

Firstly, a plurality of muscles of a healthy mouse are taken, muscle tissues are placed in a cell culture dish by using sterile ophthalmological scissors and are cut into small pieces with the diameter of 1-2mm, the cut muscle tissue pieces are visually divided into 6 parts with the same quantity for standby, the serial number is 1-6, wherein the 1 st-2 parts are used for a traditional net rubbing method experiment, the 3 rd-4 parts are used for a traditional grinding method experiment, and the 5 th-6 th parts are left to be ground by using the grinding device provided by the application to obtain single cells.

Example 1:

in this example, a single cell suspension was prepared by a conventional mesh-rubbing method, and the specific operation method comprises the following steps:

firstly, the nylon net is firmly fixed on the opening of the sterile vessel, the sterile vessel adopted by the embodiment is a small beaker, and the nylon net adopts a 300-mesh specification.

In the second step, the 1 st muscle tissue mass is soaked with the debonding reagent used in this application consisting of neutral protease at a final concentration of 2mg/mL, collagenase I at a final concentration of 0.09mg/mL, sodium chloride at a final concentration of 10mg/mL, and magnesium chloride at a final concentration of 5mM/L for 20 min.

Secondly, placing the 1 st part of the muscle tissue block after being loosened on a nylon net, repeatedly kneading the muscle tissue by using a grinding rod or other articles convenient to knead, and intermittently dropping a loosening reagent for wetting and washing while kneading until most tissues pass through the nylon net after being kneaded; during the kneading process, the muscle tissue must be kept wet, and dry kneading cannot be performed.

And thirdly, transferring the cell suspension obtained from the small beaker into a sterile centrifuge tube, and centrifuging for 5min at 1600 rpm.

And fourthly, sucking supernatant fluid to remove, washing the supernatant fluid by PBS, and then resuspending the supernatant fluid by PBS for later use, wherein the suspension fluid A is recorded.

And fifthly, repeating the steps by using the 2 nd part of the suspension to repeatedly knead, and obtaining the cell suspension, namely the suspension B.

Example 2:

in the first step, the 3 rd muscle tissue mass is soaked for 20min with a debonding reagent consisting of neutral protease at a final concentration of 2mg/mL, collagenase type I at a final concentration of 0.09mg/mL, sodium chloride at a final concentration of 10mg/mL, and magnesium chloride at a final concentration of 5 mM/L.

Secondly, filtering out the muscle tissue, transferring the muscle tissue into a grinder, and rotating a grinding rod until the tissue is homogenized visually; during the grinding, the muscle tissue should be kept moist, and if the muscle tissue is not moist, a proper amount of loosening reagent should be added in real time to continue grinding until the muscle tissue is homogenized.

Thirdly, adding sufficient physiological saline to flush the grinder, and filtering the cell suspension obtained after flushing through a 200-mesh nylon net;

fourthly, centrifuging the filtered cells, wherein the centrifugation condition is 1600rpm and 5min is obtained;

and fifthly, sucking the supernatant, removing the supernatant, washing the supernatant with PBS, and then resuspending the supernatant with PBS for later use, wherein the suspension is marked as suspension C.

And sixthly, marking the cell suspension obtained by repeating the steps on the 4 th muscle tissue block as suspension D.

Example 3:

the present embodiment is a method for preparing viable single cells by using an abrading device designed by the applicant, which is specifically shown in the accompanying drawings 1-6 of the specification, and the method specifically comprises the following steps: a method for preparing survival single cells based on grinding specifically comprises the following operation steps:

step ST100, preparing grinding materials including sterilized living biological tissue blocks, a loosening agent and a grinding device; the debonding reagent consists of neutral proteinase in the final concentration of 2mg/mL, type I proteinase in the final concentration of 0.09mg/mL, sodium chloride in the final concentration of 10mg/mL and magnesium chloride in the final concentration of 5 mM/L. It should be noted that, in this embodiment, the tissue is muscle tissue, the concentration of the relevant enzyme is measured according to the maximum concentration, and if the treated tissue is a more easily treated part, such as liver, spleen, brain, etc., the relevant measured concentration can be adaptively reduced, and even the time can be shortened, which can be determined by relatively limited experiments according to the actual tissue conditions, therefore, the application of the lysis reagent should not be limited to the different tissue parts.

ST200, cleaning the 5 th and 6 th muscle tissue blocks by using normal saline, and soaking the cleaned muscle tissue blocks in a loosening reagent for 20min, wherein the temperature environment during soaking is 18 ℃;

step ST300, placing the 5 th and 6 th soaked muscle tissue blocks and the loosening reagent into two centrifuge tubes, and adding a plurality of shearing magnetic beads into the centrifuge tubes; the shear magnetic beads comprise equal-diameter glass beads, PP beads and spherical zirconia, and the ratio of the shear magnetic beads to the shear magnetic beads is 0.8:0.8 and 1, wherein the PP beads are obtained by hot melting polypropylene and glass fibers and have the density of 0.95-1.1g/cm ³ The spheroid of (4).

ST400, placing the centrifugal tube into a grinding device for grinding, wherein the grinding device at least has back and forth oscillation of more than two moving degrees of freedom simultaneously or alternatively applied to the centrifugal tube, and the grinding time is 30min;

and step ST500, filtering the mixture of the tissue block, the cell mass and the single cell obtained in the step ST400 by using a cell screen, collecting the filtered filtrate, centrifuging again, collecting the single cell for storage, and respectively recording the obtained single cell suspension as suspension E and suspension F. In the grinding process, the 5 th muscle tissue block and the 6 th muscle tissue block are symmetrically arranged on two sides of the grinding device respectively, so that the grinding is more symmetrical.

In the preparation method, a great deal of experimental research is carried out in step ST300 to obtain the rule of the shear magnetic beads on the fragmentation and dispersion of the biological tissue, so that the shear magnetic beads are improved. The research of the applicant finds that the mode of adding the shear magnetic beads and matching with the oscillation can be applied to the conventional cell disruption, relatively ideal living single cells of biological tissues can be obtained by improving the magnetic beads and the grinding mode, and the obtained cells can keep the living state for a long time. The magnetic bead grinding method is mainly characterized in that irregular oscillation of magnetic beads is utilized, biological tissues are cut through collision to achieve the purpose of crushing, the magnetic bead grinding method is often used for extracting DNA and RNA in organelles or cells, the cells can be quickly damaged under the action of the magnetic beads, and the purpose of directional extraction can be achieved by adding related biological agents or enzymes, so that the magnetic bead crushing mode is widely applied to the field of cell crushing and DNA and RNA extraction. However, in order to bring obvious damage to cells in this way, grinding extraction using magnetic beads is hardly found in the field of viable single cells; on the basis that the applicant finds that single cells which can survive for a long time exist in the single cell suspension prepared by grinding the biological tissue blocks, in order to realize the automatic preparation of the single cells, the applicant can obtain the technical effect that the single cell rate is obviously higher than that of manual grinding by matching the developed grinding device with the improvement of shearing magnetic beads to grind the biological tissue. More importantly, the grinding process adopted by the invention can realize turnover grinding, namely, the biological tissue can realize horizontal and up-and-down irregular turnover movement under the combined action of vibration and shearing of magnetic beads in the grinding process, and can realize very uniform discrete action on the biological tissue under the frequent action of shearing of the magnetic beads. While the effect of ordinary grinding on biological tissues is relatively non-uniform with cells as a unit, there are a large number of cell clusters that cannot be ground efficiently. Therefore, the grinding mode has a very important function for obtaining more and more complete single cells, and for this reason, the applicant designs a brand new grinding device aiming at the requirement. The method comprises the following specific steps:

the polishing apparatus includes a first oscillating mechanism 2 providing a horizontal reciprocating motion and a second oscillating mechanism 7 providing a vertical reciprocating motion in step ST100 shown in fig. 1 to 4. The first oscillating mechanism 2 plays a role in shearing biological tissues by utilizing shearing magnetic bead inertia, and the second oscillating mechanism 7 plays a role in overturning the biological tissues, so that the shearing magnetic beads have more uniform and comprehensive effects on the biological tissues, shearing blind areas are eliminated, and more single cells can be obtained. In order to realize above-mentioned technological effect, the grinder that this application adopted includes first driver 5, the pivot of first driver 5 has linked firmly driving-disc 8, driving-disc 8 supports to lean on first oscillation mechanism 2 for driving-disc 8 is reciprocating motion through the level and smooth undulant lateral wall that has, first oscillation mechanism 2 slidable mounting is on the carousel 3 that uses driving-disc 8 to install as the center, 3 lower surface connections of carousel have casing 1, casing 1 with fixed connection can be dismantled to first driver 5. First vibration mechanism 2 is including sliding the setting T shape guide rail 21 in the spout, T shape guide rail 21 is close to the one end of driving-disc 8 is provided with first fixed pulley 22, and the other end fixedly connected with of T shape guide rail 21 is used for installing centrifuge tube rack 23 of centrifuging tube 24.

Preferably, the first oscillating mechanism 2 further includes a resetting mechanism 6, the resetting mechanism 6 includes a first fixing seat 61 fixedly connected to the lower surface of the rotary table 3, a second fixing seat 64 fixedly connected to the T-shaped guide rail 21, an anti-roll bar 63 is fixedly connected to the second fixing seat 64, a blind hole coaxially disposed with the anti-roll bar 63 is formed in the first fixing seat 61, a spring 62 is sleeved on the anti-roll bar 63, two ends of the spring 62 are respectively fixedly connected to the bottom of the blind hole in the first fixing seat 61 and the second fixing seat 64, and a free end of the anti-roll bar 63 extends towards the first fixing seat 61 and is inserted into the blind hole.

For the better realization realize the motion of overturning of biological tissue piece in grinding process, still include second oscillation mechanism 7, second oscillation mechanism 7 is including setting up pulley support 71 of 3 lower surfaces of carousel is provided with second fixed pulley 72 on pulley support 71, second fixed pulley 72 through with the smooth undulate surface contact promotion that T shape guide rail 21 lower surface set up T shape guide rail 21 shakes from top to bottom, there is the clearance between the upper surface of guide rail 21 and the spout inner wall on the carousel 3, install throughout and promote in the clearance the downward reed 4 of hugging closely with carousel 3 of track 21.

Example 4:

in this example, the suspension a and the suspension B were mixed, and the mixture was shaken up and recorded as a net rubbing group; mixing the suspension C and the suspension D, shaking up and recording as a grinding group; mix suspension E and suspension F and shake well as record this application. The pictures of the cells obtained are shown in FIGS. 7-9.

The number of single cells was measured by a flow cytometer from the rubbed group, the ground group and the cell suspension obtained in the present application, and the number and ratio of viable cells were measured by a trypan blue staining method to obtain the parameters shown in table 1 below.

Serial number	Group of	Number of single cells/cell	Cell mass/number	Number of living cells/cell	Average viable cell ratio/%)
						1	Grinding group	8.23×10 ⁴	5.67×10 ²	2.63×10 ⁴	31.9％
2	Net rubbing set	1.2×10 ⁵	4.2×10 ²	0.77×10 ⁵	35.0％
						3	This application	0.87×10 ⁶	2.03×10 ²	0.49×10 ⁶	56.3％

Note: the counting operation and the live cell counting operation shown in table 1 above were as follows:

rubbing the net group, the grinding group and the cell suspension obtained by the method, wherein 10ul of cell suspension is taken from each group, the cell suspension is injected from one side of a cell counting plate, then the cell suspension is placed under a microscope for counting, and finally the number of cells is calculated;

viable cell counting method:

taking 10ul of cell suspension, adding 10ul of 0.4% trypan blue cell dye solution, mixing uniformly, taking 10ul of mixed liquid, transferring to a cell counting plate, calculating the number of cells which are not dyed into blue, and finally obtaining the number of living cells.

As can be seen from Table 1, the yield of single cells obtained by the conventional web rubbing and grinding methods is very low, the survival rate of single cells in the obtained single cell suspension is less than 40%, which does not include dead single cells after standing for a certain period of time, and the number of the obtained survival single cells is much less if the single cell suspension is cultured for a certain period of time. The application is due to the addition of special shear beads. Comprises glass beads with equal diameters and PP beads, wherein the PP beads are obtained by hot melting polypropylene and glass fibers and have the density of 0.95-1.1g/cm ³ The spheroid of (a); the density of the PP beads is equivalent to that of the solution, so that the PP beads can be uniformly distributed in the solution in the shaking process and uniformly and gently act on muscle tissues, and the absolute value of the cell yield of the obtained single-cell suspension is several times that of the cell yield of the single-cell suspension in the traditional manual operation modeAlmost one order of magnitude higher, and the survival rate is over half, reaching 56.3%. Although the yield and survival rate are much lower than those of the tissue ablator disclosed in the patent publication No. CN109609500B developed by the applicant in the application of extracting viable single cells (the ablator can achieve more than 90% survival rate by ablating tissues through parameter optimization setting), the survival rate which can be half as high as that of the existing physical mode is remarkably improved; meanwhile, the difficulty of obtaining single cells of muscle tissues is obviously higher than that of single cells of spleen and liver tissues; therefore, the applicant believes that the method for obtaining single cells by pure physical action provided by the application has substantial characteristics and remarkable progress under the known technology obtained by the existing pure physical way. In examining this application, the technical effect, i.e., absolute value of the number of single-cell yields and the viable cell rate, should be strictly compared with physically prepared single-cell suspensions, and should not be compared with non-physically prepared single-cell suspensions; in this regard, the research and development team of the applicant also repeatedly utilizes a non-physical mode for preparation, such as an ultrasonic ablation mode, the technical effect of the ultrasonic ablation mode is completely different from that of the physical mode in the same order of magnitude, but the ultrasonic ablation equipment is high in price and different from the practicability and the universality of a pure physical mode in the same day, so that the method provided by the application can meet the requirements of most laboratories, automation and high efficiency can be realized and the yield is more stable compared with the conventional manual homogenization.

The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing viable single cells based on grinding is characterized by comprising the following operation steps:

step ST100, preparing grinding materials including sterilized living biological tissue blocks, a loosening agent and a grinding device; the debonding reagent consists of neutral protease with the final concentration of 2mg/mL, type I collagen protease with the final concentration of 0.09mg/mL, sodium chloride with the final concentration of 6-10mg/mL and magnesium chloride with the final concentration of 2-5 mM/L; the grinding device comprises a first oscillating mechanism (2) providing horizontal reciprocating motion and a second oscillating mechanism (7) providing vertical reciprocating motion; the grinding device comprises a first driver (5), a driving disc (8) is fixedly connected to a rotating shaft of the first driver (5), the driving disc (8) abuts against a first oscillating mechanism (2) through a smooth fluctuating side wall to reciprocate relative to the driving disc (8), the first oscillating mechanism (2) is slidably mounted on a turntable (3) mounted by taking the driving disc (8) as the center, a shell (1) is connected to the lower surface of the turntable (3), and the shell (1) is detachably and fixedly connected with the first driver (5);

ST200, cutting the sterilized and alive biological tissue blocks into a plurality of small blocks with the length not more than 2mm by using sterile ophthalmic scissors, then cleaning the small blocks by using normal saline, and soaking the cleaned biological tissue blocks in a debonding reagent for 20-30min, wherein the temperature environment during the soaking is 18-24 ℃;

step ST300, placing the soaked biological tissue block and the loosening reagent in a centrifugal tube, and adding a plurality of shearing magnetic beads into the centrifugal tube; the shear magnetic beads comprise glass beads with equal diameters and PP beads which are mixed according to the proportion of 0.5-0.8 ³ The spheroid of (a); the shear magnetic beads further comprise spherical zirconia of equal diameter;

step ST500, filtering the tissue block, the cell mass and the single cell mixture obtained in the step ST400 by using a cell screen, collecting the filtered filtrate, centrifuging, collecting the single cell, and storing for later use;

first vibrate mechanism (2) including sliding T shape guide rail (21) that sets up in the spout, T shape guide rail (21) are close to the one end of driving-disc (8) is provided with first fixed pulley (22), and the other end fixedly connected with of T shape guide rail (21) is used for installing centrifuge tube rack (23) of centrifuging tube (24).

2. The method for preparing viable single cells based on grinding as claimed in claim 1, wherein the first oscillating mechanism (2) further comprises a reset mechanism (6), the reset mechanism (6) comprises a first fixing seat (61) fixedly connected to the lower surface of the rotary disk (3), a second fixing seat (64) fixedly connected to the T-shaped guide rail (21), an anti-roll bar (63) is fixedly connected to the second fixing seat (64), the first fixing seat (61) is provided with a blind hole coaxially arranged with the anti-roll bar (63), the anti-roll bar (63) is sleeved with a spring (62), two ends of the spring (62) are respectively fixedly connected with the bottom of the blind hole on the first fixing seat (61) and the second fixing seat (64), and the free end of the anti-roll bar (63) extends towards the first fixing seat (61) and is inserted into the blind hole.

3. The grinding-based method for preparing viable single cells according to claim 2, further comprising a second oscillating mechanism (7), wherein the second oscillating mechanism (7) comprises a pulley bracket (71) arranged on the lower surface of the rotary disc (3), a second fixed pulley (72) is arranged on the pulley bracket (71), the second fixed pulley (72) pushes the T-shaped guide rail (21) to vibrate up and down through contacting with a smooth undulating surface (211) arranged on the lower surface of the T-shaped guide rail (21), a gap exists between the upper surface of the T-shaped guide rail (21) and the inner wall of the chute on the rotary disc (3), and a pressure spring piece (4) which always pushes the T-shaped guide rail (21) to downwards and tightly attach to the rotary disc (3) is arranged in the gap.