CN115354018A - Human fetal membrane tissue single cell rapid dissociation kit, dissociation method and application - Google Patents
Human fetal membrane tissue single cell rapid dissociation kit, dissociation method and application Download PDFInfo
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Abstract
The invention relates to a kit for rapidly dissociating human fetal membrane tissue single cells, a dissociation method and application. The kit for the single cell rapid dissociation of the human fetal membrane tissue comprises the following components: enzyme I, enzyme II, combination enzyme, dilution buffer, neutralization buffer, wash buffer, and 3 x red blood cell lysis buffer. The dissociation method provided by the invention firstly separates the fetal membrane tissue into the amniotic membrane and the chorion-decidua, and then adopts a mode of combining single enzyme and combined enzyme to carry out synchronous and independent enzymolysis, so that compared with other enzymolysis modes (comparative examples 1 to 5), the dissociation method has the advantages that the efficiency is obviously improved, the dissociation time is shortened to 70min from 6h, and the cell viability, the viable cell concentration and the agglomeration rate are obviously superior. The experimental cost is greatly reduced, and meanwhile, the accuracy and the efficiency of the experiment are improved.
Description
Technical Field
The invention belongs to the field of biological medicines, and particularly relates to a human fetal membrane tissue single cell rapid dissociation kit, a dissociation method and application.
Background
The fetal membrane, which is located in the lining of the uterine cavity during pregnancy and provides mechanical, immunological, endocrine and antimicrobial functions during pregnancy, can act as a barrier between the fetal placenta and the maternal compartment and play an important role in protecting the fetus and transmitting maternal-fetal signals related to the onset of labor. The fetal membrane structural framework consists of the amniotic membrane (the innermost layer of the amniotic cavity) and the chorion (the fetal tissue associated with the maternal decidua). The amnion is in direct contact with amniotic fluid, which is the main reaction person of amnion cavity change; the chorion is closely adjacent to the decidua of the mother and is responsible for maintaining immune tolerance at the interface between mother and fetus. During normal pregnancy, the fetal membranes undergo a systemic remodeling process by balancing the locally controlled inflammatory environment to accommodate fetal growth and development and amniotic fluid biochemical environmental changes.
However, the research of the prior art on the structure and function of the fetal membrane at the molecular mechanism level is still very limited, and in any single cell research, the process of preparing the single cell suspension is very important and directly related to whether the subsequent experiment can be smoothly carried out, and the process is also the problem to be solved by the single cell sequencing and flow cell sorting technology.
In the process of preparing the suspension, a dissociation kit is indispensable, however, no report of the dissociation kit for preparing single cell suspension by human fetal membrane tissue is found at present. Since the composition of the biological matrix of various tissues and organs of human body is greatly different, different tissue types for dissociating and preparing single cells need to be searched for corresponding components of the dissociation kit. Meanwhile, the prior art has the problems of high cell death rate, more cell fragments, unclean cell background or high agglomeration rate caused by damaged cell aggregation in the preparation of suspension. It affects the final data quality, whether single cell sequencing data or purity of flow/magnetic cell sorting, which is very likely to lead to misinterpretations by researchers.
Therefore, the technical scheme of the invention is provided.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a human fetal membrane tissue single cell rapid dissociation kit, a dissociation method and application.
The invention provides a human fetal membrane tissue single cell rapid dissociation kit, which comprises the following components: enzyme I, enzyme II, combined enzyme, dilution buffer, neutralization buffer, washing buffer and 3 Xerythrocyte lysis buffer; wherein:
the enzyme I is used for improving the tissue penetrating ability and is neutral protease;
the enzyme II is used to dissociate epithelial cell types, which is trypsin;
the combined enzyme is used for dissociating chorion-decidua and comprises type IV collagen hydrolase, neutral protease and deoxyribonuclease I;
the dilution buffer solution is used for adjusting enzyme and other buffer solutions to the optimal working solution concentration, and is a D-HBSS balanced salt solution, and the D-HBSS balanced salt solution comprises NaCl, KCl and Na 2 HPO 4 、Na 2 HPO 4 ·12H 2 O, D-Glucose and sterile deionized water;
the neutralization buffer is used for inhibiting enzyme activity and comprises fetal calf serum and D-HBSS balanced salt solution;
the washing buffer is used for eluting cell debris and comprises bovine serum albumin and a D-HBSS balanced salt solution;
the 3 × erythrocyte lysis buffer is used for removing residual erythrocytes, including NH 4 Cl、KHCO 3 、Na 2 EDTA and sterile deionized water. It should be emphasized that the residue may be determined according to the actual residueThe amount of the remaining red blood cells is 1 to 3 Xthe red blood cell lysis buffer prepared by a dilution buffer.
Preferably, the human fetal membrane tissue single cell rapid dissociation kit comprises the following components in concentration:
the neutral protease is 1.2 to 2.4U/mL;
the trypsin accounts for 0.25 to 0.5wt.%;
the type IV collagen hydrolase is 1 to 3mg/mL, the neutral protease is 2.4U/mL, and the deoxyribonuclease I is 25 mu g/mL;
NaCl is 0.4mg/mL, KCl is 8mg/mL, na 2 HPO 4 0.35mg/mL of the Na 2 HPO 4 ·12H 2 O is 0.06mg/mL, and D-Glucose is 1mg/mL;
the fetal bovine serum is 10wt.%;
1 to 4wt.% of bovine serum albumin;
the NH 4 Cl of 150mM, said KHCO 3 Is 10mM, said Na 2 EDTA was 0.1mM.
Based on the same technical idea, another aspect of the present invention is to provide a dissociation method, including the following steps:
(I) Pretreatment:
transferring the fetal membrane tissue to a culture dish to remove surface residual blood clots, and further separating the fetal membrane into an amniotic membrane part and a chorion-decidua part;
(II) single cell dissociation of amniotic tissue:
(1) Cutting amniotic tissue into pieces, transferring the pieces into an EP tube filled with enzyme I, sealing, and transferring the EP tube into a water bath for standing for primary digestion;
(2) Taking out the predigested amnion, transferring the amnion into a centrifuge tube filled with an enzyme II, standing in a water bath for secondary digestion, taking out the remaining amnion tissue for later use after the secondary digestion is finished, and obtaining a digestive juice;
(3) Adding a neutralization buffer solution into the digestive juice, uniformly mixing, and filtering to remove residual microtissue blocks to obtain a cell suspension;
(4) Centrifuging the cell suspension to remove supernatant to obtain cell sediment; adding a washing buffer solution into the cell sediment, centrifuging, collecting the cell sediment again, and resuspending by adopting the washing buffer solution for later use;
(5) Transferring the residual amniotic tissue taken out in the step (2) into a centrifuge tube, adding a combined enzyme for third digestion, adding a neutralization buffer solution after complete digestion, mixing uniformly, filtering, centrifuging, and collecting cell precipitates;
(6) Adding a washing buffer solution into the cell sediment collected in the step (5), uniformly mixing, centrifuging to remove supernatant, collecting the cell sediment again, and then resuspending by using the washing buffer solution for later use;
(III) Single cell dissociation of chorion-decidua tissue (step II performed simultaneously):
(S1) shearing and crushing chorion-decidua tissues, transferring the chorion-decidua tissues into a centrifugal tube filled with enzyme I, putting the centrifugal tube into a water bath for incubation, removing supernate after incubation, and adding combined enzyme;
(S2) putting the centrifugal tube into the water bath again, standing and incubating, adding the enzyme II after the incubation is finished, and continuing the water bath after the enzyme II is uniformly mixed;
(S3) adding a neutralization buffer solution into a centrifugal tube after complete digestion, filtering and centrifuging in sequence after uniform mixing, and collecting cell precipitates;
(S4) resuspending the cell sediment by using a washing buffer solution, centrifuging, collecting the cell sediment again, and resuspending by using the washing buffer solution for later use;
(IV) lysis of erythrocytes:
(SS 1) mixing the cell suspensions obtained in the steps (4), (6) and (S4), and centrifuging to obtain cell precipitates;
(SS 2) resuspending the cell pellet with a washing buffer, and adding 3 × erythrocyte lysis buffer;
(SS 3) centrifugally collecting cell sediment after red blood cracking is finished, cleaning the cell sediment by adopting a washing buffer solution, removing supernatant, re-suspending cells by using the washing buffer solution, and filtering to obtain the fetal membrane single cell suspension.
Preferably, in step (2), the first digestion is performed by: standing and digesting for 5 to 15 min at 37 ℃, taking out from the water bath every 5 to 10min, and shaking and mixing uniformly.
Preferably, in step (2), the second digestion is performed by: standing and digesting for 15 to 25 min at 37 ℃, taking out from the water bath every 5 to 10min, and shaking and mixing uniformly.
Preferably, in step (5), the third digestion mode is: standing and digesting for 20 to 30min at 37 ℃, taking out from the water bath every 5 to 10min, and shaking and mixing uniformly.
Based on the same technical concept, the invention also provides an application of the single cell suspension in single cell sequencing and flow/magnetic cell sorting.
The application one is as follows: single cell suspension preparation for single cell sequencing
The types, states and interactions of cells vary greatly between various tissues in humans. The single cell RNA sequencing (snRNA-seq) technology provides a method for observing gene expression at the single cell level, and can better research tissues and different types of cells existing in the tissues. This technique can be used to:
(1) Studying which types of cells are present in a tissue at all;
(2) Identifying unknown or rare cell types or states;
(3) Elucidating changes in gene expression during the differentiation process or over time and state;
(4) Finding genes that are differentially expressed in a particular type of cell under different conditions (e.g., drug-loading group and disease group);
(5) Changes in gene expression between cell types are explored while incorporating spatial, regulatory and/or protein information.
The technical process of single cell sequencing comprises the following steps: single cell suspension preparation, single cell separation and library construction, sequencing, data analysis and visual interpretation. The dissociation of tissue single cells is to prepare high-activity and high-quality single cell suspension from fresh tissues, is the first step of single cell transcriptome sequencing (scRNA-seq), and is also a key step for restricting the smooth proceeding of single cell experiments and successfully acquiring sequencing data.
The application II comprises the following steps: single cell suspension preparation for flow/magnetic cell sorting
With the continuous development of cell therapy and gene therapy technologies, the related research of cells has gradually become a research hotspot in the current medical and even whole life science fields, cell culture has become an important technology in scientific research, and flow/magnetic cell sorting and purification have become key problems to obtain purer target cells. This important technique can be used:
(1) Stem cell tissue engineering is under investigation;
(2) Cell proliferation, activity and apoptosis studies;
(3) The occurrence, development and outcome research of disease cells such as inflammatory cells, tumor cells and the like;
(4) Gene expression and expression regulation studies;
(5) Research on intracellular signal transduction and interaction between biological macromolecules;
(6) Basic and pathogenic mechanism researches of various pathogens such as viruses and bacteria;
(7) Research on pharmacological efficacy and drug development;
(8) Transplantation and cell therapy studies;
(9) The effect of biological materials on cellular structure, activity and function, etc.
Flow/magnetic cell sorting separates a cell from a multicellular sample. The cells are labeled with antibodies, and the antibodies are fluorescently labeled or linked to immunomagnetic beads or others, and the cells are isolated by the characteristics of fluorescence and immunomagnetic beads. The dissociation of the tissue single cell is to prepare a high-activity and high-quality single cell suspension from a fresh tissue, is the first step of separating single type cells from composite type tissue cells by a flow cell separation technology, and is also a key step for restricting the smooth operation of a cell separation experiment and successfully obtaining high-purity single type cells.
The invention has the beneficial effects that:
the invention provides a technology for rapidly dissociating fetal membrane tissues, which comprises the steps of firstly, dissecting and separating human fetal membrane tissues into an amniotic membrane part and a chorionic decidua part, and then, carrying out synchronous and independent enzymolysis in a mode of combining a single enzyme and a combined enzyme, wherein compared with other enzymolysis modes (comparative examples 1 to 5), the efficiency is obviously improved, the dissociation time is shortened to 70min from 6h, and the cell activity rate, the viable cell concentration and the agglomeration rate are obviously superior; the experimental cost is greatly reduced, and meanwhile, the accuracy and the efficiency of the experiment are improved.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the prior art descriptions will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a photograph of the cell suspension obtained in the example in the bright field.
FIG. 2 is a photograph of the fluorescence field of the cell suspension obtained in the example.
FIG. 3 is a bright field image of the cell suspension obtained in comparative example 1.
FIG. 4 is a photograph of the fluorescence field of the cell suspension obtained in comparative example 1.
FIG. 5 is a photograph of the bright field of the cell suspension obtained in comparative example 2.
FIG. 6 is a photograph of the fluorescence field of the cell suspension obtained in comparative example 2.
FIG. 7 is a bright field image of the cell suspension obtained in comparative example 3.
FIG. 8 is a photograph of the fluorescence field of the cell suspension obtained in comparative example 3.
FIG. 9 is a bright field picture of the cell suspension obtained in comparative example 4.
FIG. 10 is a photograph of the fluorescence field of the cell suspension obtained in comparative example 4.
FIG. 11 is a bright field image of the cell suspension obtained in comparative example 5.
FIG. 12 is a photograph of the fluorescence field of the cell suspension obtained in comparative example 6.
FIG. 13 is a statistical chart of cell viability in examples and comparative examples 1 to 5.
FIG. 14 is a statistical graph of the viable cell concentrations of examples and comparative examples 1 to 5.
FIG. 15 is a statistical chart of the agglomeration rates in examples and comparative examples 1 to 5.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the examples given herein without any inventive step, are within the scope of the present invention.
Examples
This example provides a method for dissociation of human fetal membrane tissue single cells, wherein the required material equipment is shown in table 1, and the components and concentrations of the required kit are shown in table 2.
TABLE 1 materials equipment
TABLE 2 kit Components and concentrations
The dissociation method comprises the following steps:
(I) Pretreatment:
(1) Storing and transporting the tissue in a pre-cooled tissue protection solution;
(2) The freshly delivered fetal membrane tissue is transferred to a petri dish containing a wash buffer composition, and blood clots are carefully cleared without damaging decidua tissue;
(3) Holding a scalpel, taking down the full-layer tissue block, putting the full-layer tissue block into a clean culture dish, holding an ophthalmic forceps to separate the amnion from the chorion-decidua tissue, respectively transferring the amnion and the chorion-decidua tissue into the clean culture dish, and carrying out the next operation.
(II) single cell dissociation of amniotic tissue:
(S1) shearing amniotic membrane tissue by holding an ophthalmic scissors;
(S2) transferring the cut tissue into a 1.5ml EP tube filled with the enzyme I, and sealing the EP tube by using a sealing film;
(S3) placing the EP tube into a water bath, standing and digesting for 10min at 37 ℃, taking out the EP tube from the water bath every 5min, slightly shaking by hand, after digestion is completed, holding the ophthalmic forceps to fish out the amnion, placing the amnion into a centrifugal tube filled with 1mL of enzyme II, re-suspending tissue particles by shaking the centrifugal tube, standing for 20min at 37 ℃, taking out the amnion from the water bath every 5min and slightly shaking by hand, taking out the amnion tissue for later use after completion, and obtaining a digestive juice;
(S4) adding a precooled neutralization buffer solution into the digestive juice, blowing, beating and uniformly mixing, and filtering through a cell sieve to remove agglomerated cells and tissue blocks;
(S5) centrifuging the filtered cell suspension in a centrifugal machine for 5min, and removing a supernatant;
(S6) adding a pre-cooled washing buffer solution into the cell sediment, uniformly blowing and stirring, centrifuging the cell suspension in a centrifuge for 5min, collecting, then re-suspending by using the pre-cooled washing buffer solution, and standing on ice for later use;
(S7) fishing out the amniotic membrane tissue in the step (3) by holding the ophthalmological forceps, transferring the amniotic membrane tissue into a clean centrifugal tube, adding the combined enzyme into the centrifugal tube, continuing to digest the amniotic membrane tissue at 37 ℃ for 30min, taking out the amniotic membrane tissue from the water bath every 5min, and slightly shaking the amniotic membrane tissue by hand;
(S8) after complete digestion, adding a precooled neutralization buffer solution into the centrifuge tube, and blowing, beating and uniformly mixing; filtering with cell sieve, centrifuging in a centrifuge for 5min, and collecting cells;
(S9) removing the supernatant, using a precooled washing buffer solution to resuspend the cell sediment, centrifuging the cell suspension in a centrifuge for 5min, collecting the cell sediment, using a precooled washing buffer solution to resuspend, and placing on ice for standby.
(III) single cell dissociation of chorion-decidual tissue:
(SS 1) shearing chorion-decidua tissue with ophthalmic scissors;
(SS 2) transferring the tissue to a centrifuge tube containing enzyme I, incubating at 37 ℃ for 10min while taking out from the water bath every 5min and shaking gently by hand;
(SS 3) after incubation, removing the supernatant and adding thereto the combinatorial enzyme;
(SS 4) transferring the centrifuge tube into a water bath kettle at 37 ℃, standing and incubating for 30min, taking out the centrifuge tube from the water bath every 5min, and lightly shaking the centrifuge tube by hands;
(SS 5) adding the enzyme II into the centrifuge tube, blowing, beating and uniformly mixing, and continuing water bath for 20min;
(SS 6) after complete digestion, adding a precooled neutralization buffer solution into a centrifuge tube, and blowing, beating and uniformly mixing; filtering with cell sieve, centrifuging in centrifuge for 5min, and collecting cells;
(SS 7) removing the supernatant, resuspending the cell pellet using a pre-cooled wash buffer, centrifuging the cell suspension in a centrifuge for 5min, collecting the cell pellet and resuspending using a wash buffer.
(IV) erythrocyte lysis:
(SSS 1) mixing the cells obtained in the step (S6), the step (S9) and the step (SS 7) uniformly, and centrifuging for 5min in a centrifuge; resuspending the obtained cell pellet with a precooled washing buffer, slowly adding 3 × erythrocyte lysis buffer (if the amount of erythrocytes is less, diluting the erythrocyte lysis buffer to 1 × with a dilution buffer, and then using the diluted erythrocyte lysis buffer), and placing the erythrocyte lysis buffer on ice for 5min to lyse erythrocytes;
(SSS 2) after the completion of the erythrolysis, centrifuging the cells in a centrifuge for 5min to collect the cells, and washing the cell precipitate once with a washing buffer;
(SSS 3) removing the supernatant, then using a washing buffer solution to resuspend the cells, and filtering by using a cell sieve to obtain the total single cell suspension of the fetal membranes.
Finally, the cells are stained by an AO/PI dye and counted. The cell suspension obtained was subjected to quality control using a cell counter, and the results showed: the concentration of the living cells is 184 ten thousand/mL; FIG. 1 shows that bright field pictures show that the background is clean, the number of cell fragments and residual red blood cells is small, and the cell morphology types are rich; from FIG. 2, it can be seen that the cell viability in the fluorescence field is 92.9% and the clumping rate is 3.9% (based on the AO/PI count). Meanwhile, the method also shows that the operation is performed by selecting a fresh fetal membrane-decidua sample as much as possible, the fetal membrane-decidua in-vitro time is long, cells are easy to die, and the final experimental effect is influenced.
It is emphasized that the inventor, in combination with the pathophysiological techniques of fetal membranes, considers that fetal membranes are a relatively dense tissue rich in complex matrix components such as collagen, laminin, proteoglycan, etc. and simultaneously present various types of cells, and a complex enzymatic method using trypsin, neutral protease and collagenase in combination is the most preferable solution.
Among them, trypsin exists in the digestive system of most vertebrates, has the function of hydrolyzing and digesting proteins, and for tissues and cells, the trypsin has the function of hydrolyzing proteins among cells so as to achieve the function of dispersing cells, and in consideration of different actions, reactions and tolerance of different tissues or cells to the trypsin, conditions, namely concentration, time and temperature, when the trypsin is used for dissociation are higher, so that the cells are prevented from being damaged due to overdigestion; meanwhile, if the digestion time is too short, it is difficult to sufficiently break intercellular junctions, resulting in insufficient cell dispersion and a high agglomeration rate.
Collagenase, also known as collagen hydrolase, specifically hydrolyzes collagen peptide bonds without causing other types of tissue proteins, and is therefore particularly suitable for collagen-rich tissue types such as skin, colorectal, and the like, as well as fetal membranes; considering that the actual collagenase has 6 types, namely collagenase I, collagenase II, collagenase III, collagenase IV, collagenase V and collagenase VI, and the fetal membrane tissue is mainly collagen VI, the collagenase IV can be selected for tissue dissociation.
The neutral protease is an endonuclease, can hydrolyze macromolecular protein into amino acid under the conditions of proper temperature and pH, is suitable for the hydrolysis of various animal proteins, and needs balanced salt solution containing fetal calf serum for the termination of digestion.
Therefore, the inventor establishes a mild dissociation scheme based on the combination of the complex enzyme and the single enzyme of the specificity, the distribution dissociation and the time dependence of the fetal membrane tissues, and provides reliable guarantee for obtaining the fetal membrane single cell suspension and fully guaranteeing the cell activity.
Comparative example 1
Comparative example 1 provides a dissociation method of human fetal membrane tissue single cells, which is different from the examples in that the present comparative example performs enzymatic hydrolysis using only neutral protease (single enzyme). Finally, AO/PI dye is adopted for dyeing and counting, a bright field picture is shown in figure 3, and a fluorescence field picture is shown in figure 4.
Since the neutral protease action site is in basement membrane type IV collagen and fibronectin, the neutral protease action site is only limited to loosening tissues by destroying hemidesmosome and improving the tissue penetrating capacity, and the number of cells obtained after digestion is small. This theoretical analysis is consistent with the results shown in the figures.
Comparative example 2
Comparative example 2 provides a method for dissociation of single cells of human fetal membrane tissue, which is different from the examples in that the present comparative example performs enzymatic hydrolysis using only trypsin (single enzyme). Finally, AO/PI dye is adopted for staining and counting, the bright field picture is shown in figure 5, and the fluorescence field picture is shown in figure 6.
Since trypsin mainly acts on connexins adhered to the surface of epithelial-type cells, but since the outer layer of fetal membrane tissue is covered with a large amount of collagen, trypsin cannot effectively penetrate the tissue and can not digest interstitial-type cells in the collagen layer, only a small amount of epithelial-type cells are obtained after digestion, and a large amount of cells are easily killed after long-term digestion. This theoretical analysis is consistent with the results shown in the figures.
Comparative example 3
Comparative example 3 provides a method for dissociation of human fetal membrane tissue single cells, which is different from the examples in that the comparative example performs enzymolysis using collagenase type iv (single enzyme) only. Finally, AO/PI dye is adopted for staining and counting, a bright field picture is shown in figure 7, and a fluorescence field picture is shown in figure 8.
Because collagenase IV mainly acts on interstitial type cells rich in collagen, and epithelial cell types are insensitive to collagenase IV, only a small amount of interstitial type cells are obtained after digestion by the single enzyme. This theoretical analysis is consistent with the results shown in the figures.
In addition, it should be emphasized that dnase only acts on free DNA caused by cell dissociation to reduce cell death caused by DNA entanglement, and thus cannot be used alone as a subject of single cell dissociation.
Comparative example 4
Comparative example 4 provides a method for dissociation of human fetal membrane tissue single cells, which is different from the examples in that the comparative example performs enzymolysis only using a combination enzyme consisting of collagenase type iv and trypsin. Finally, AO/PI dye is adopted for staining and counting, a bright field picture is shown in figure 9, and a fluorescence field picture is shown in figure 10.
Due to the lack of trypsin to dissociate epithelial cell types, digestion with this combination of enzymes only yields a large number of mesenchymal cells, resulting in a lower number of epithelial cells. This theoretical analysis is consistent with the results shown in the figures.
Comparative example 5
Comparative example 5 provides a method for dissociation of human fetal membrane tissue single cell, which is different from the examples in that the present comparative example uses neutral protease as a single enzyme, uses collagenase type iv and trypsin as combined enzymes, and performs enzymolysis in a manner that the combined enzymes are added simultaneously with the single enzyme. Finally, AO/PI dye is adopted for staining and counting, a bright field picture is shown in figure 11, and a fluorescence field picture is shown in figure 12.
On one hand, the single enzyme trypsin and the combined enzyme IV collagenase and neutral protease are added simultaneously to perform single cell dissociation, which easily causes epithelial cell death due to the action of the trypsin for a long time along with the dissociation of the combined enzyme for a long time, and on the other hand, the dissociation of mesenchymal cells is insufficient according to the dissociation time of the trypsin. This theoretical analysis is consistent with the results shown in the figures.
The cell viability, viable cell concentration, and agglomeration rate of examples and comparative examples 1 to 5 were counted, and the results are shown in fig. 13, 14, and 15, respectively. As can be seen from fig. 13 to 15, only using a single enzyme results in insufficient dissociation of some cells, the concentration of the viable cells of the finally obtained cell suspension is low, the cell types are single, the cell aggregation rate is high, the cell ratio does not conform to the real ratio of the cells in the tissue, and simultaneously, adding all types of proteases to treat the cells greatly reduces the cell aggregation rate, and the cell aggregation rate is also at a high level.
Meanwhile, the invention selects four enzymes, namely neutral protease, type IV collagen hydrolase, trypsin and deoxyribonuclease I from a plurality of enzymes, and compared with other disclosed invention patent enzyme combinations, the invention better accords with the histological characteristics of fetal membranes, and the types and concentrations of the screened and optimized mixed enzymes are more targeted and efficient. The method provides a good tool for obtaining single cells from tissue types which are difficult to dissociate and contain more collagen and more types of cells, and is also a technical method which can successfully dissociate fetal membranes and meet single cell sequencing conditions and is developed for the first time.
The enzymolysis method combining the single enzyme and the combined enzyme is an innovation after considering the tissue structure and the cell anatomical distribution of the fetal membranes. After optimizing the conditions such as the optimal mixture ratio and digestion time (as described in the examples), the optimized enzyme concentration ratio is more consistent with the histological characteristics of fetal membranes than other published enzyme combinations of the invention patents. Obtaining cell suspension which is relatively in line with the standard of single cell sequencing on a computer, and compared with the similar products which only use a single enzyme method, the method improves the obtaining quantity of different types of cells, so that the tissue cell composition integrity in the obtained single cell suspension is higher; meanwhile, the combined enzyme form is only used in a key step, so that the cost and the time are saved compared with a method of simultaneously using a plurality of collagenases in a broad spectrum, and more importantly, the cell death caused by over digestion of the cells which are not tolerant to the traditional mixed enzyme method treatment is effectively reduced.
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily think of the changes or substitutions within the technical scope of the present invention, and shall cover the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (8)
1. The kit for the single cell rapid dissociation of the human fetal membrane tissue is characterized by comprising the following components: enzyme I, enzyme II, combined enzyme, dilution buffer, neutralization buffer, washing buffer and 3 Xerythrocyte lysis buffer; wherein:
the enzyme I is neutral protease;
the enzyme II is trypsin;
the combined enzyme comprises type IV collagen hydrolase, neutral protease and deoxyribonuclease I;
the dilution buffer solution is a D-HBSS balanced salt solution, and the D-HBSS balanced salt solution comprises NaCl, KCl and Na 2 HPO 4 、Na 2 HPO 4 ·12H 2 O, D-Glucose and sterile deionized water;
the neutralization buffer solution comprises fetal calf serum and a D-HBSS balanced salt solution;
the washing buffer solution comprises bovine serum albumin and a D-HBSS balanced salt solution;
the 3 × erythrocyte lysis buffer comprises NH 4 Cl、KHCO 3 、Na 2 EDTA and sterile deionized water.
2. The kit for rapid dissociation of human fetal membrane tissue single cells as claimed in claim 1, which comprises the following components in concentration:
the concentration of the neutral protease is 1.2 to 2.4U/mL;
the concentration of the trypsin is 0.25 to 0.5wt.%;
in the combined enzyme, the concentration of the type IV collagen hydrolase is 1 to 3mg/mL, the concentration of the neutral protease is 2.4U/mL, and the concentration of the deoxyribonuclease I is 25 mug/mL;
the concentration of NaCl is 0.4mg/mL, the concentration of KCl is 8mg/mL, and the Na is 2 HPO 4 Has a concentration of 0.35mg/mL of the Na 2 HPO 4 ·12H 2 The concentration of O is 0.06mg/mL, and the concentration of D-Glucose is 1mg/mL;
the concentration of fetal bovine serum is 10wt.%;
the concentration of the bovine serum albumin is 1 to 4wt.%;
the NH 4 The concentration of Cl is 150mM, the KHCO 3 In a concentration of 10mM, said Na 2 The concentration of EDTA was 0.1mM.
3. A dissociation method for dissociating human fetal membrane tissue by using the kit for rapid dissociation of single cells of human fetal membrane tissue as claimed in claim 1, the dissociation method comprising the steps of:
i, pretreatment:
transferring the fetal membrane tissue to a culture dish to remove surface residual blood clots, and further separating the fetal membrane into an amniotic membrane part and a chorion-decidua part;
II, single cell dissociation of amniotic membrane tissue:
s1, cutting amniotic tissues into pieces, transferring the pieces into an EP tube filled with enzyme I, sealing, and transferring the EP tube into a water bath for standing for primary digestion;
s2, taking out the pre-digested amnion, transferring the amnion into a centrifuge tube filled with an enzyme II, standing in a water bath for secondary digestion, taking out the remaining amnion tissue for later use after the secondary digestion is finished, and obtaining a digestive juice;
s3, adding a neutralization buffer solution into the digestive juice, uniformly mixing, and filtering to remove residual micro-tissue blocks to obtain a cell suspension;
s4, centrifuging the cell suspension to remove supernatant to obtain cell sediment; adding a washing buffer solution into the cell sediment, centrifuging, collecting the cell sediment again, and resuspending the cell sediment by adopting the washing buffer solution for later use;
s5, transferring the remaining amniotic membrane tissue taken out in the step S2 into a centrifuge tube, adding a combined enzyme for third digestion, adding a neutralization buffer solution after complete digestion, mixing uniformly, filtering, centrifuging and collecting cell precipitates;
s6, adding a washing buffer solution into the cell sediment collected in the step S5, uniformly mixing, centrifuging to remove supernatant, collecting the cell sediment again, and then resuspending by using the washing buffer solution for later use;
III single cell dissociation of chorion-decidual tissue:
SS1, shearing the chorion-decidua tissue, transferring the chorion-decidua tissue into a centrifugal tube filled with enzyme I, placing the centrifugal tube in a water bath for incubation, removing supernatant after incubation, and adding the combined enzyme;
SS2, putting the centrifuge tube into the water bath again, standing and incubating, adding the enzyme II after the incubation is finished, and continuing the water bath after the enzyme II is uniformly mixed;
SS3, adding a neutralization buffer solution into a centrifugal tube after complete digestion, filtering and centrifuging in sequence after uniform mixing, and collecting cell precipitates;
SS4, using a washing buffer solution to resuspend the cell sediment, centrifuging, collecting the cell sediment again, using the washing buffer solution to resuspend, and finishing for later use;
IV erythrocyte lysis:
SSS1, mixing the cell suspensions obtained in the steps S4, S6 and SS4, and centrifuging to obtain cell precipitates;
SSS2, adopting washing buffer solution to resuspend the cell sediment, and adding 3 Xerythrocyte lysis buffer solution;
and centrifuging to collect cell sediment after SSS3 and red blood burst are finished, cleaning the cell sediment by adopting a washing buffer solution, re-suspending cells by using the washing buffer solution after supernatant is removed, and filtering to obtain the fetal membrane single cell suspension.
4. The dissociation method according to claim 3, wherein in step S2, the first digestion is performed by: standing and digesting at 37 ℃ for 5 to 15 min, taking out from the water bath every 5 to 10min, and shaking and mixing uniformly.
5. The dissociation method according to claim 3, wherein in step S2, the second digestion is performed by: standing and digesting for 15 to 25 min at 37 ℃, taking out from the water bath every 5 to 10min, and shaking and mixing uniformly.
6. The dissociation method according to claim 3, wherein in step S5, the third digestion is performed by: standing and digesting at 37 ℃ for 20 to 30min, taking out from the water bath every 5 to 10min, and shaking and mixing uniformly.
7. A single cell suspension obtained by the dissociation method according to any one of claims 3 to 6.
8. Use of the single cell suspension of claim 7 for single cell sequencing, flow/magnetic cell sorting.
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