CN113265371B - Preparation method of efficient human kidney single cell suspension - Google Patents
Preparation method of efficient human kidney single cell suspension Download PDFInfo
- Publication number
- CN113265371B CN113265371B CN202110533402.8A CN202110533402A CN113265371B CN 113265371 B CN113265371 B CN 113265371B CN 202110533402 A CN202110533402 A CN 202110533402A CN 113265371 B CN113265371 B CN 113265371B
- Authority
- CN
- China
- Prior art keywords
- kidney
- cell suspension
- single cell
- cell
- sample
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0684—Cells of the urinary tract or kidneys
- C12N5/0686—Kidney cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
- C12N2509/10—Mechanical dissociation
Abstract
The invention provides a high-efficiency preparation method of a human kidney single cell suspension. According to the method, after the kidney tissue sample is sheared in the EP tube, the kidney tissue sample is digested and incubated by FCMase with specific concentration, and after intermittent uniform mixing, the kidney tissue sample is resuspended and purified by a DMEM culture medium, so that the obtained kidney single cell has complete morphology, good dispersity, a large number of kidney single cells and high activity.
Description
Technical Field
The invention belongs to the field of cell biology, and particularly relates to a preparation method of a high-efficiency human kidney single-cell suspension.
Background
With the development and innovation of scRNA-seq research and flow cytometry experiments, the application of flow cytometry analysis technology and single-cell transcriptome in kidney diseases is becoming wider and wider, including sorting cells with specific functions, identifying disease types, finding novel heterogeneous cell subsets, accurately finding cell levels, finding cell groups and genes with specific functions, evaluating new therapeutic targets of diseases, evaluating therapeutic effects of drugs and the like. Single cell preparation is a key part of experiments such as single cell transcriptome, flow cytometry and primary cell culture.
However, the differentiation degree of kidney tissues is high, the preparation of single cell suspension is difficult, and the digestion is usually performed by adopting an enzyme digestion method mainly comprising collagenase, so that in the clinical application process, the puncture sample size is small, and the enzyme digestion of the kidney into single cells with high quality and large quantity by adopting a proper and efficient method is continuously explored.
In view of this, the present invention has been proposed.
Disclosure of Invention
The invention aims to seek a preparation method of high-efficiency human kidney single cell suspension. In order to achieve the above purpose, the invention provides a high-efficiency preparation method of single cell suspension of human kidney by exploring the operation method, enzyme concentration, enzyme action time, centrifugation rate and cell purification method, and specifically provides the following technical scheme.
The invention firstly provides a preparation method of a high-efficiency kidney single-cell suspension, which comprises the following steps:
1) Sample processing: taking a kidney in-vitro tissue sample, shearing, cleaning with PBS, and centrifuging to remove the supernatant;
2) And (3) enzyme digestion: adding 0.111-0.125 mg/. Mu.L of tissue digestive enzyme FCMase, incubating at 37deg.C for 10-20min, mixing for 5-8min, and stopping digestion with DMEM complete medium;
3) And (3) a heavy suspension purification step: the resulting cell suspension was centrifuged to remove the supernatant, and purified after the DMEM medium was resuspended.
Further, the step 2) is as follows: the tissue digestion enzyme FCMase was added at a final concentration of 0.125 mg/. Mu.L, incubated at 37℃for 20min, mixed well every 5min, and the digestion was stopped with DMEM complete medium.
Further, the step 1) is as follows: the kidney isolated tissue sample was taken in an EP centrifuge tube, cut into small pieces with scissors, washed with PBS, centrifuged at 300 Xg for 5-8min, and the supernatant removed.
Further, the step 3) is as follows: the resulting single cell suspension was filtered through a 70 μm cell sieve, centrifuged at 300 Xg for 5-8min, the supernatant removed, resuspended in 3mL of DMEM medium and purified.
Further, the purification in the step 3) is separation by density gradient, magnetic bead or flow separation;
in some preferred embodiments, the purification is flow sorting; in some more preferred embodiments, the flow sorting is: 1mL of the flow-on-machine staining solution was added to the suspension, 10. Mu.L of 7-AAD was added thereto, and the mixture was incubated for 10min and sorted by a flow cytometer.
Further, no red blood cell lysate is added after digestion in step 2).
Further, the kidney single cells are derived from human kidney single cells.
Further, the kidney sample is derived from a kidney puncture sample.
In some preferred embodiments, when the kidney sample is derived from a kidney puncture sample, the step 1) is: the kidney puncture sample was placed in a 5mL centrifuge tube, washed with 3mL PBS, centrifuged at 300 Xg for 5min, the supernatant removed, the puncture sample was sheared with scissors, washed again with PBS, and centrifuged at 300 Xg for 5min to remove the supernatant.
The invention also provides an application of FCMase in preparing kidney single cell suspension, wherein the application is as follows: adding a tissue digestive enzyme FCMase with a final concentration of 0.111-0.125 mg/mu L into the kidney puncture sample;
further, digestion and incubation at 37 ℃ for 10-20min are included, and the mixture is uniformly mixed every 5-8 min.
In some preferred embodiments, this involves adding the tissue digesting enzyme FCMase at a concentration of 0.125 mg/. Mu.L, incubating at 37℃for 20min, mixing every 5min, and stopping digestion with DMEM complete medium.
Further, before adding the tissue digestion enzyme fcase, the method further comprises the step of sample treatment: taking a kidney sample, shearing, putting into PBS for cleaning, and centrifuging to remove supernatant;
in some embodiments, the sample processing step: the kidney samples were taken in centrifuge tubes, cut into small pieces with scissors, washed with PBS, centrifuged at 300 Xg for 5-8min, and the supernatant removed.
Further, after adding the tissue digestion enzyme fcase, the method further comprises a step of re-suspension purification: the resulting cell suspension was centrifuged to remove the supernatant, and purified after the DMEM medium was resuspended.
In some embodiments, the resuspension purification step is: the resulting cell suspension was filtered through a 70 μm cell sieve, centrifuged at 300 Xg for 5min, the supernatant removed, resuspended in 3mL of DMEM medium and purified.
In some preferred embodiments, the purification in the resuspension purification step is by density gradient separation, magnetic beads, or flow sorting; in some more preferred embodiments, the purification is flow sorting, in particular: 1mL of the flow-on-machine staining solution was added to the suspension, 10. Mu.L of 7-AAD was added thereto, and the mixture was incubated for 10min and sorted by a flow cytometer.
Further, the enzyme digestion is followed by no addition of red blood cell lysate.
Further, the kidney single cells are derived from human kidney single cells.
Further, the kidney sample is derived from a kidney puncture sample.
The invention also provides a kit for preparing the kidney single-cell suspension, which comprises components such as digestive enzyme FCMase, PBS, DMEM complete medium and the like.
Further, the concentration of the digestive enzyme FCMase is 0.111-0.125 mg/. Mu.L, more preferably 0.125 mg/. Mu.L.
In some embodiments, the digestive enzyme fcase is available from HyperCyte company under the trademark HC0146.
Compared with the prior art, the invention has at least the following advantages:
1) The invention optimizes and establishes a high-efficiency preparation method of the kidney single cell suspension, and the cell yield and the activity of the digested cells are high, and 10mg is usually 10mg, namely the number of the extractable cells of a puncture kidney tissue sample is (2-3) multiplied by 10 6 A plurality of; the number of single cells of the kidney-penetrating tissue after the frozen tissue is treated by the method is (5-8) multiplied by 10 5 。
2) The enzyme used in the invention can separate more single cells in a short time, and then the single cells are removed by density gradient separation, magnetic beads or flow separation, so as to obtain the mononuclear cells with higher activity.
3) According to the invention, in terms of operation methods, samples are sheared in the EP tube, so that the loss of sample quantity in the operation process of the samples is avoided.
4) The invention cleans the tissue twice with PBS before enzyme digestion, thereby reducing the impurity in the cell suspension.
5) In many documents, after tissue digestion, red blood cell lysate is added to lyse red blood cells in suspension cells, but the invention finds that the red blood cell lysate has larger damage to cells, the dead cell number is increased sharply, and cell debris is also increased; if the sample size is small, the addition of red blood cell lysate is not recommended for treatment.
6) Too high a centrifugation rate will increase the cell death rate and too low an eccentricity, and the cell debris in the prepared cell suspension will increase, and the invention is just suitable for using a specific centrifugation of 300 Xg for 5 min.
7) According to the invention, the kidney sample is incubated by adding the enzyme, and the sample and the enzyme can be contacted with each other by adding the enzyme into the kidney sample and uniformly mixing the sample and the enzyme every 5 minutes, so that more single cells can be obtained by full digestion.
8) The method can separate target cells from tissues with high yield and high purity to obtain a mixture of various cells, and can obtain a sufficient quantity of mononuclear cells for clinical puncture diagnosis. The preparation of single cell samples in the aspect of accurate detection is also overcome, and single cell transcriptome analysis can be used for comprehensively knowing the single cell level change in the disease process, so that the process of accurate medical treatment in the kidney field can be promoted.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 effect of different feed ratios on single cell viability;
FIG. 2 effect of different incubation times on single cell viability;
FIG. 3 effect of different treatments on single cell viability;
FIG. 4 effect of different cell purification treatments on single cell viability;
FIG. 5 shows the effect of different cell purification treatments on cell mass yield;
FIG. 6 effect of treatments 1-3 on cell mass yield;
FIG. 7 effect of treatment mode 4 on cell mass yield;
FIG. 8 effect of cell eccentricity on survival of the preparation single cells;
FIG. 9 effect of cell eccentricity on impurities in the preparation of cell suspensions;
FIG. 10 isolated human kidney single cell morphology×400;
FIG. 11 shows a single cell suspension flow cytometry of human kidney;
FIG. 12 is a graph showing the status statistics of isolated human kidney single cells.
Detailed Description
The following description of the embodiments of the present invention will be made apparent and fully in view of the accompanying drawings, in which some, but not all embodiments of the invention are shown. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The following terms or definitions are provided solely to aid in the understanding of the invention. These definitions should not be construed to have a scope less than understood by those skilled in the art.
Unless defined otherwise hereinafter, all technical and scientific terms used in the detailed description of the invention are intended to be identical to what is commonly understood by one of ordinary skill in the art. While the following terms are believed to be well understood by those skilled in the art, the following definitions are set forth to better explain the present invention.
As used herein, the terms "comprising," "including," "having," "containing," or "involving" are inclusive or open-ended and do not exclude additional unrecited elements or method steps. The term "consisting of …" is considered to be a preferred embodiment of the term "comprising". If a certain group is defined below to contain at least a certain number of embodiments, this should also be understood to disclose a group that preferably consists of only these embodiments.
The indefinite or definite article "a" or "an" when used in reference to a singular noun includes a plural of that noun.
The terms "about" and "substantially" in this invention mean the range of accuracy that one skilled in the art can understand yet still guarantee the technical effect of the features in question. The term generally means a deviation of + -10%, preferably + -5%, from the indicated value.
Furthermore, the terms first, second, third, (a), (b), (c), and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.
The following are specific examples.
The invention is practical material and main reagent
Human kidney tissue was from Shanxi province people's hospitals and DMEM basal medium was purchased from Hyclone company; 1 XPBS was purchased from Boshide bioengineering Co., ltd; fcase tissue digests were purchased from HC0146, hyperCyte corporation; RS-I Medium is available from AQIX company; magLive dead cell removal kit was purchased from qdsophere corporation; percoll cell isolates were purchased from Pharmacia; PI is available from YEASEN company; 7-AAD is available from BD company.
The experimental method is as follows:
the isolated tissue was adventitially de-fatted in a petri dish and fresh samples were preserved in tissue preservation solution (AQIX). A portion of the tissue mass was stored for 30 minutes using FRS, 1ml of CS10 frozen stock was added, and stored at-80℃overnight with liquid nitrogen. A part of tissue blocks are put into a 50mL centrifuge tube, can be cut into small blocks by scissors, and are respectively weighed; a penetrating tissue sample may also be taken with the penetrating needle. The resulting sample was placed in a 5mL centrifuge tube, washed with 3mL of LPBS, centrifuged at 300 Xg for 5min, the supernatant removed, the puncture sample was sheared with scissors, washed with PBS, and the supernatant removed by centrifugation. Adding digestive enzyme, incubating at 37deg.C for 20min, mixing every 5min, stopping digestion with DMEM complete medium, filtering the obtained cell suspension with 70 μm cell sieve, centrifuging at 300×g for 5min, removing supernatant, and re-suspending with 3mL of DMEM medium.
The detection index and statistical analysis are as follows: making the puncture tissue sample into 3mL of cell suspension, taking 1mL of cell suspension, centrifuging to remove supernatant, re-suspending cell sediment by using 1mL of flow staining solution, adding 2uL of pI labeled cells, and detecting the cell activity of the obtained mononuclear cells by using an up-flow cytometer. Counting the prepared single cells by using a cell counting plate under a microscope, dying by using 0.4% trypan blue, adding the single cells into the cell suspension according to the solution ratio of 1:9, reacting for 3min, and recording the death rate of the cells; cell morphology was observed with an inverted phase contrast microscope. Statistical and mapping of data was performed using Graphpad prism 6 software.
Example 1 Effect of enzyme selection and enzyme concentration on preparation of Single cell suspensions
The early-stage experimental screening shows that the digestive enzyme FCMase can be used for digesting kidney samples, and the effect is obviously better than that of the existing digestive enzyme system. The FCMase digestive juice contains unique protease and collagenase activities, and can be rapidly and mildly applied to kidney samples.
Regarding selection of the enzyme concentration, kidney tissue samples were taken, the tissues were minced, the mass was weighed, fcase enzyme was added at final concentrations of 0.167, 0.143, 0.125, 0.111 mg/. Mu.l, respectively, and incubated at 37 ℃ for 20min, and mixed once every 5 min. Other steps are as above, single cell suspensions are prepared.
As shown in the results of FIG. 1, when the sample masses were the same, the single cell viability was affected differently by the addition of different volumes of digestive enzymes, wherein the single cell suspension prepared at FCMase enzyme concentrations of 0.111-0.125 mg/. Mu.L, especially 0.125 mg/. Mu.L, had the highest activity ratio.
Example 2 Effect of digestion time of digestive enzymes on preparation of Single cell suspensions
Taking kidney in-vitro tissue samples, weighing, adding digestive enzyme according to the concentration of 0.125 mg/mu L feed liquid, namely adding about 10mg of 1 puncture tissue sample, adding 80 mu L FCMase enzyme, incubating at 37 ℃ for respectively digesting for 10min, 20min and 30min, and uniformly mixing every 5 min. Other steps are as above, single cell suspensions are prepared.
As shown in FIG. 2, the effect of different incubation times of the same enzyme on single cell survival rate tends to increase and decrease, when FCMase enzyme is added, the single cell suspension prepared by incubating for 10-20min, especially 20min at 37 ℃ has the highest activity ratio.
EXAMPLE 3 Effect of erythrocyte lysate on preparation of Single cell suspension
After preparing single cell suspension from fresh human kidney in vitro tissues, repeating three tubes, wherein one group is not added with erythrocyte lysate, the other group is centrifuged, 3mLACK Lying is added, incubation is carried out on ice for 5min, supernatant is removed by centrifugation, PBS is added for resuspension, the prepared cell suspension is prepared, and the influence of the two groups on the survival rate of the single cell suspension is compared and observed.
As shown in the results of FIG. 3, the addition of the red blood cell lysate caused a doubling of the number of dead cells in the single cells, and the addition of the red blood cell lysate to perform red blood cell lysis removal of the single cell suspension was not suitable in the case of a smaller fresh tissue sample. It can be seen that the lysis of erythrocytes, which is used for the extraction of single cells in blood samples, is not suitable for use in tissues.
EXAMPLE 4 Effect of different purification methods on preparation of Single cell suspensions
Preparing single cell suspension from fresh human kidney in vitro tissue, repeating three tubes as a group, wherein the group is taken as a control group without purification treatment, adding 50 mu L/mL of dead cell-removing magnetic beads in an experimental group 1, standing for 5min, incubating for 10min on a magnetic rack, washing cells twice with PBS, and re-suspending; the magnetic beads of purified DNA were added to the experimental group 2, reacted for 5min, incubated on a magnetic rack for 10min, the liquid was collected, centrifuged, and resuspended in PBS. 3mL of 30% percoll working solution is added into the experimental group 3, 1mL of cell suspension is placed on the upper layer of 30% percoll solution, the mixture is stood for 5min,400 Xg is centrifuged for 40min, the upper layer liquid is removed after layering, the cells are washed twice by PBS, and the cells are resuspended; treatment mode 4 the frozen puncture tissue was taken into EP tubes, three tubes were repeated in one set, one set was used as a control, and after the other set was made into single cell suspension, 1mL of flow-type on-machine staining solution was added, 10 μl of 7-AAD was added for 10min incubation, and sorting was performed by flow cytometry. The effect of these four treatments on cell number and viability of single cell suspensions was observed.
As can be seen from fig. 4-7, the cell viability of the treated group was improved by 20% -30% and the proportion of cell clusters was reduced by 5% -7% compared to the control group, and the cell viability was obtained by the purification treatment: flow sorting > magnetic bead sorting > percoll density gradient centrifugation sorting > magnetic bead optimization of purified DNA. The effect of the purification and the flow separation of the magnetic beads for removing dead cells in the treatment group is very little; compared with a single cell suspension obtained by a fresh tissue sample, the single cell survival rate of the frozen puncture tissue sample is lower. As can be seen from FIGS. 5 and 6, the magnetic beads for purifying DNA reduce the majority of the cell mass, and separate into monocytes, and it is presumed that some of the cell mass is bound together by the oligonucleotide chains in the cell debris.
Example 5 Effect of cell eccentricity on preparation of Single cell suspension
In the process of preparing single-cell suspension, multiple times of centrifugation are needed, and different eccentricity has a certain influence on the quality of single cells prepared from the single-cell suspension. The above experimental procedure was followed, wherein a group was treated with 100 Xg of centrifugal force on the tissue and cell suspension; the other group was treated with a centrifugal force of 300 Xg and a single cell suspension was prepared, and the effect of both groups on the survival rate of the single cell suspension and the effect of the single cell state were observed in comparison.
As shown by the results in FIG. 8, decreasing the eccentricity results in an insignificant decrease in the number of dead cells in a single cell. As is clear from FIG. 9, although the eccentricity of 100 Xg increased the proportion of living cells, the amount of impurities in the cell suspension was observed under a microscope, and the amount of cell debris was also relatively large, and in the overall view, the centrifugation of 300 Xg was selected to be more preferable.
In summary, examples 1-5, establish the optimal method of the invention: for human kidney samples, the mass is weighed, the samples are sheared by scissors, washed by PBS, the supernatant is removed by centrifugation, and the tissue digestion enzyme FCMase is added according to the proportion of 0.125 mg/mu L, and if the samples are puncture samples, 80 mu L of digestive enzyme can be used for tissue digestion. Incubation was carried out at 37℃for 20min, after every 5min, digestion was stopped with DMEM complete medium, the resulting cell suspension was filtered through a 70 μm cell sieve, centrifuged at 300 Xg for 5min, the supernatant removed, resuspended in 3mL of DMEM medium and purified.
The cell digested by the method has high yield and good activity, and the number of the cell which can be extracted by one puncture kidney tissue sample is generally 10mg (2-3) multiplied by 10 6 A plurality of; the number of single cells of the kidney-penetrating tissue after the frozen tissue is treated by the method is (5-8) multiplied by 10 5 。
EXAMPLE 6 preparation of cell suspension of puncture tissue sample and analysis of cell Activity
The preparation method of the single cell of the human kidney cell based on the established optimal method comprises the following specific steps: a. taking kidney tissue of a patient; removing adventitia fat from the tissue in a culture dish; c. immersing the fresh sample in the tissue preservation solution; d. taking a puncture tissue sample by using a puncture needle, placing the puncture tissue sample into a 5mL centrifuge tube, cleaning by using 3mLPBS, centrifuging for 5min at 300 Xg, and removing supernatant; e. the puncture sample is sheared by scissors, put into PBS for cleaning and centrifuged to remove the supernatant. f. Adding digestive enzyme, incubating at 37deg.C for 20min, and stopping digestion with DMEM complete medium; g. filtering with 70um cell sieve, centrifuging for 5min, and removing supernatant; h. the 7-AAD-labeled cells were resuspended in 3mL of DMEM medium and negative single cells were flow sorted.
Observing cell bodies through a microscope, and performing FCMase digestion under a microscope to obtain single cells of the human kidney, wherein the single cells are various in morphology, different in size and good in dispersity; the background is cleaner, the impurities and cell fragments are less, and the cell clusters are less. At 37℃5% CO 2 The morphology of the cells after one day of culture in the cell incubator is shown in FIG. 10. As shown in FIG. 11, the percentage of viable cells in the single cell suspension produced was 80%, and flow cytometry examined the single cell suspension for an active large number of kidney mononuclear cells. FIG. 12 is a statistical diagram of the state of isolated human kidney single cells, the human kidney single cells obtained by the method have lower aggregation number, the aggregation rate of living cells is 7-14%, and the aggregation rate of dead cells is 2-7%; wherein the cell mass composed of two cells can reach 14%, and the cell mass composed of three cells is about 5%. The average diameter of living cells is 9.678 +/-0.237 mu m, the average diameter of dead cells is 12.225 +/-0.507 mu m, and the monocytes with larger volumes in human kidneys are more easily inactivated.
Therefore, the method is simple to operate, has high efficiency, high yield and other effects, even exceeds the expected effect, and is suitable for popularization and use aiming at a small amount of puncture clinical samples, and the human kidney cells prepared by the method have large single cell number and extremely high activity.
The foregoing descriptions of specific exemplary embodiments of the present invention are presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application to thereby enable one skilled in the art to make and utilize the invention in various exemplary embodiments and with various modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.
Claims (4)
1. The preparation method of the efficient kidney single cell suspension is characterized by comprising the following steps of:
1) Sample processing: taking a kidney in-vitro tissue sample, shearing the kidney in an EP tube, putting the cut kidney in PBS for cleaning, and centrifuging to remove supernatant;
2) And (3) enzyme digestion: adding the tissue digestive enzyme FCMase with the final concentration of 0.111-0.125 mg/mu L of the feed liquid, incubating for 10-20min at 37 ℃, uniformly mixing every 5-8min, and stopping digestion by the DMEM complete culture medium to obtain a cell suspension;
3) And (3) a heavy suspension purification step: filtering and centrifuging the obtained cell suspension to remove supernatant, and purifying after the DMEM culture medium is resuspended;
the kidney single cell is derived from a human kidney single cell;
the kidney ex vivo tissue sample is derived from a kidney ex vivo puncture sample;
the tissue digestive enzyme fcase is selected from digestive enzymes of HyperCyte company No. HC0146;
the step 1) is as follows: taking a kidney in-vitro tissue sample, placing the kidney in an EP centrifuge tube, cutting the kidney into small pieces by scissors, cleaning the kidney by PBS, centrifuging the kidney for 5 to 8 minutes at 300 Xg, and removing supernatant;
the red blood cell lysate is not added after digestion in the step 2);
the step 3) is as follows: filtering the obtained cell suspension with 70 μm cell sieve, centrifuging at 300×g for 5-8min, removing supernatant, re-suspending with DMEM culture medium, and purifying; the purification is by flow sorting.
2. The method for preparing a single cell suspension of kidney according to claim 1, wherein the step 2) comprises: the tissue digestion enzyme FCMase was added at a final concentration of 0.125 mg/. Mu.L, incubated at 37℃for 20min, mixed once every 5min, and the digestion was stopped with DMEM complete medium.
3. The method for preparing a single cell suspension of kidney according to claim 1, wherein the step 1) is: the kidney puncture sample was placed in a 5mLEP tube, washed with 3mLPBS, centrifuged at 300 Xg for 5min, the supernatant removed, the puncture sample was cut into small pieces with scissors, washed again with PBS, and centrifuged at 300 Xg for 5min to remove the supernatant.
4. The method of preparing a renal single cell suspension of claim 1, wherein the flow sorting is: 1mL of the flow-on-machine staining solution was added to the suspension, 10. Mu.L of 7-AAD was added thereto, and the mixture was incubated for 10min and sorted by a flow cytometer.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110533402.8A CN113265371B (en) | 2021-05-17 | 2021-05-17 | Preparation method of efficient human kidney single cell suspension |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110533402.8A CN113265371B (en) | 2021-05-17 | 2021-05-17 | Preparation method of efficient human kidney single cell suspension |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113265371A CN113265371A (en) | 2021-08-17 |
| CN113265371B true CN113265371B (en) | 2023-09-15 |
Family
ID=77231128
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110533402.8A Active CN113265371B (en) | 2021-05-17 | 2021-05-17 | Preparation method of efficient human kidney single cell suspension |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113265371B (en) |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2469481A1 (en) * | 1993-10-08 | 1995-04-27 | The Regents Of The University Of Michigan | Growing renal tubule cells and ex vivo renal tubule tissue system |
| CN102286429A (en) * | 2011-07-20 | 2011-12-21 | 中国人民解放军第二军医大学 | Sarcomatoid renal cell carcinoma cell line from Han people and preparation method thereof |
| WO2016186506A1 (en) * | 2015-05-20 | 2016-11-24 | Academisch Ziekenhuis Leiden H.O.D.N. Lumc | Collections of primary kidney cells, method of isolation and uses thereof |
| CN106929468A (en) * | 2017-03-21 | 2017-07-07 | 西南大学 | A kind of preparation method of fish single cell suspension |
| CN107748130A (en) * | 2017-10-16 | 2018-03-02 | 上海市普陀区中心医院 | A kind of preparation of animal hearts single cell suspension and detection method |
| CN112251398A (en) * | 2020-11-12 | 2021-01-22 | 中国农业大学 | Separation and extraction method of primary parenchymal hepatocytes and application thereof |
| CN112725262A (en) * | 2021-02-25 | 2021-04-30 | 武汉博尔夫生物科技有限公司 | Method for extracting primary cells of suckling mouse kidney |
| CN113403295A (en) * | 2021-06-07 | 2021-09-17 | 山西省人民医院 | Digestive enzyme for preparing human kidney tissue single cell suspension and application |
| CN113684174A (en) * | 2021-08-26 | 2021-11-23 | 山西省人民医院 | Preparation method of human kidney podocyte |
-
2021
- 2021-05-17 CN CN202110533402.8A patent/CN113265371B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2469481A1 (en) * | 1993-10-08 | 1995-04-27 | The Regents Of The University Of Michigan | Growing renal tubule cells and ex vivo renal tubule tissue system |
| CN102286429A (en) * | 2011-07-20 | 2011-12-21 | 中国人民解放军第二军医大学 | Sarcomatoid renal cell carcinoma cell line from Han people and preparation method thereof |
| WO2016186506A1 (en) * | 2015-05-20 | 2016-11-24 | Academisch Ziekenhuis Leiden H.O.D.N. Lumc | Collections of primary kidney cells, method of isolation and uses thereof |
| CN106929468A (en) * | 2017-03-21 | 2017-07-07 | 西南大学 | A kind of preparation method of fish single cell suspension |
| CN107748130A (en) * | 2017-10-16 | 2018-03-02 | 上海市普陀区中心医院 | A kind of preparation of animal hearts single cell suspension and detection method |
| CN112251398A (en) * | 2020-11-12 | 2021-01-22 | 中国农业大学 | Separation and extraction method of primary parenchymal hepatocytes and application thereof |
| CN112725262A (en) * | 2021-02-25 | 2021-04-30 | 武汉博尔夫生物科技有限公司 | Method for extracting primary cells of suckling mouse kidney |
| CN113403295A (en) * | 2021-06-07 | 2021-09-17 | 山西省人民医院 | Digestive enzyme for preparing human kidney tissue single cell suspension and application |
| CN113684174A (en) * | 2021-08-26 | 2021-11-23 | 山西省人民医院 | Preparation method of human kidney podocyte |
Non-Patent Citations (9)
| Title |
|---|
| Isolation of Viable Single Cells With High Yield and Purity Using a Small Amount of Human Kidney Tissue Biopsy;Hasnaa Yaigoub等;《Frontiers in cell and developmental biology》;20220510;第1-9页 * |
| Nanda等.Comparative study on enzymatic and explant methdo in establishing primary culture from different cultivable cells of india major carp,cirrhinus mrigala.《Asian Journal of Animal and Veterinary Advances》.2014,第281-291页. * |
| 人肾癌干细胞的分离，鉴定及其特异性microRNA的筛选和功能研究;胡峻珲;《中国博士学位论文电子期刊网》;20150715(第7期);全文 * |
| 剪碎法及匀浆法制备兔肌肉VX2肿瘤单细胞悬液的方法比较;高斌;贺克武;李嘉嘉;;中国组织工程研究与临床康复(第41期);全文 * |
| 单细胞RNA测序在肾脏疾病研究中的应用;陈麒麟;《肾脏病与透析肾脏移植杂志》;20191025;第28卷(第4期);第355-359页 * |
| 孕期咖啡因暴露所致子代大鼠糖代谢功能改变的宫内起源及可遗传性;寇皓;《中国博士学位论文电子期刊网医药卫生科技辑》;20180115(第1期);第1-125页 * |
| 改良的大鼠近端肾小管上皮细胞分离纯化方法;王光兰等;《中华肾脏病杂志》;全文 * |
| 肾组织单细胞悬液制备方法的比较研究;夏安周等;《徐州医学院学报》;20040125(第01期);第1.3节，第2节 * |
| 肾脏固有树突状细胞在肾缺血-再灌注期间的变化;药晨等;《器官移植》;20160929(第05期);第42-46页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113265371A (en) | 2021-08-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP3702459B1 (en) | Method for improving fetal hemoglobin expression | |
| CN107299082B (en) | Method for separating placenta mesenchymal cells from tissues and culturing into mesenchymal stem cells | |
| EP1181524B1 (en) | Method for enriching or depleting tumour cells obtained from a body fluid and kit suitable for this purpose | |
| CN112481195B (en) | Pulmonary artery tissue single cell suspension and preparation method and application thereof | |
| WO2011069117A1 (en) | Method of isolation of stem cell populations from peripheral blood using sized-based separation (elutriation) | |
| CN112226406A (en) | Preparation method of human perivascular adipose tissue single cell suspension | |
| JP2022511935A (en) | Cell isolation for use in automated bioreactors | |
| CN113186156A (en) | Method for efficiently obtaining single cells in adipose tissue | |
| CN113025567A (en) | Separation method of intervertebral disc single cells | |
| CN111088222B (en) | Preparation method of single-cell suspension of adipose tissue | |
| CN111073858A (en) | YAP1 gene modified mesenchymal stem cell and preparation method thereof | |
| CN112111456B (en) | Circulating tumor cell separation and enrichment kit | |
| CN113265371B (en) | Preparation method of efficient human kidney single cell suspension | |
| CN111575236B (en) | Preparation method of active single cell suspension of human liver cancer tissue and liver tissue | |
| CN106267425A (en) | AIDS immunoadsorption therapy instrument | |
| CN115678852A (en) | Method for high-purity enrichment of single tumor cells in tumor tissue | |
| CN113980814B (en) | Composition for rapidly lysing peripheral red blood cells and application thereof | |
| US20230116841A1 (en) | Method for obtaining endometrial mesenchymal stem cells from human menstrual blood | |
| CN114703120A (en) | Method for separating animal nervous system blood vessel smooth muscle cell single cell | |
| CN108660108A (en) | A kind of method enhancing umbilical cord mesenchymal stem cells immunoregulation capability | |
| CN108949686B (en) | Method for obtaining hematopoietic stem cells from placenta in hypoxic environment | |
| CN111518745A (en) | Preparation method and detection method of human parathyroid single cell suspension | |
| Yaigoub et al. | Isolation of viable single cells with high yield and purity using a small amount of human kidney tissue biopsy | |
| CN108103018A (en) | A kind of method for being enriched with human blood MNCs | |
| CN110628715B (en) | Method for in vitro amplification of natural killer cells and natural killer T cells and pharmaceutical composition thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |