WO2022252753A1 - 一种骨骼组织单细胞悬液的制备方法 - Google Patents

一种骨骼组织单细胞悬液的制备方法 Download PDF

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WO2022252753A1
WO2022252753A1 PCT/CN2022/081254 CN2022081254W WO2022252753A1 WO 2022252753 A1 WO2022252753 A1 WO 2022252753A1 CN 2022081254 W CN2022081254 W CN 2022081254W WO 2022252753 A1 WO2022252753 A1 WO 2022252753A1
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孙子奎
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上海派森诺生物科技有限公司
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  • the invention relates to the technical field of gene detection, in particular to a preparation method of bone tissue single cell suspension.
  • Single-cell sequencing technology In recent years, with the rapid development of technology in the field of biological research, single-cell sequencing technology has emerged as the times require. A new technology. Single-cell sequencing can reveal the gene structure and gene expression of a single cell, classify and compare cells, and reflect the heterogeneity between cells. It plays an important role in the fields of tumors, developmental biology, immunology, and microbiology.
  • tissue single-cell suspensions with good cell viability, which restricts research work on tissue cell culture, establishment of cell lines, and functional gene verification. Therefore, the efficient preparation of tissue single-cell suspension is helpful to establish and optimize the in vitro culture technology of adult tissue cells, provide important reference materials for the establishment of cell lines, and have important theoretical and practical aspects in the research fields of functional genes, genetic breeding, etc. significance.
  • the invention provides a method for preparing bone tissue single cell suspension.
  • the scheme of the present invention is:
  • a preparation method of bone tissue single cell suspension comprising the following steps:
  • the culture medium includes serum-free medium and complete medium, prepare cell cleaning solution, and prepare mixed enzyme dissociation stock solution;
  • Tissue pretreatment transfer the bone tissue and tissue preservation solution to a 15ml test tube, absorb the tissue protection solution; absorb the cell cleaning solution and gently rinse the tissue 1-2 times, and remove the cell washing after standing for 3 minutes each time liquid;
  • step 7) To measure the yield of viable cells, absorb the AO/PI dye solution and the same volume of the target tissue single cell suspension in step 6) to fully mix, then pipette and fill it into the pool in the counting plate, and measure the viable cells by the cell counter. cell ratio.
  • the basal medium of 99ml and the penicillin-streptomycin mixed solution of 1ml are contained in the described serum-free medium of every 100ml
  • the FBS of 10ml and the FBS of 10ml are contained in the described medium of every 100ml.
  • 1ml of penicillin-streptomycin mixed solution, the basal medium is ⁇ -MEM medium.
  • the cell cleaning solution is a DPBS-5% FBS solution
  • the DPBS-5% FBS solution contains 95ml DPBS solution and 5ml FBS solution.
  • the mixed enzyme dissociation stock solution includes lyophilized powder of type II collagenase, lyophilized powder of type I collagenase and serum-free basal medium, and 30 mg of type II collagenase is added to every 30 ml of serum-free basal medium.
  • Collagenase lyophilized powder and 30 mg type I collagenase lyophilized powder were used to prepare a mixed enzyme dissociation stock solution, and the serum-free basal medium was ⁇ -MEM medium.
  • the cell filter in step 5) is a 40 ⁇ m cell filter.
  • the method for obtaining the single-cell suspension of the target tissue in the step 6) is as follows:
  • PBS-5% FBS resuspension count 1200rpm, 6min after centrifugation, discard the supernatant; add 250-500 ⁇ l DPBS-5%FBS solution, gently pipette, resuspend and mix; draw 10 ⁇ l resuspended cell suspension and mix with 10 ⁇ l of AO/PI staining solution was mixed and poured into the counting plate, and the original concentration of the cell suspension was obtained after detection by the cell counter.
  • the invention also discloses a bone tissue single cell suspension.
  • the invention also discloses the application of bone tissue single cell suspension in single cell sequencing.
  • a method for preparing a bone tissue single-cell suspension 1) preparing reagents and culture medium, the culture medium includes serum-free medium and complete medium, preparing cell cleaning solution, and preparing mixed enzymatic hydrolysis From the original solution; 2) Tissue pretreatment, transfer the bone tissue and tissue preservation solution to a 15ml test tube, absorb the tissue protection solution; absorb the cell cleaning solution and gently rinse the tissue 1-2 times, and let it stand for 3 minutes each time Remove the cell cleaning solution; 3) Initially digest and dissociate the tissue, add the 3-5ml mixed enzyme dissociation stock solution; digest and incubate in a 37°C water bath for 25 minutes; Gently pipette and mix well, no small pieces of tissue can be seen with the naked eye, and transfer to a 50ml test tube; 4) The tissue is thoroughly digested for the second time, centrifuged to remove the supernatant, centrifuged at 1200rpm, 5min; resuspended for incubation,
  • the method for preparing bone tissue single-cell suspension of the present invention can realize low-cost, fast and high-efficiency acquisition of bone tissue single-cell suspension, and ensure that the dissociated cells maintain good vitality, and can be applied to a wide range of animal bones
  • the dissociation, separation and purification of cells and the establishment of cell lines provide important reference materials for animal single cell, gene function and genetic breeding research.
  • Fig. 1 is the preparation flowchart of the present invention
  • Fig. 2 is a diagram of the quality inspection results (AO/PI) of the bone tissue single cell suspension in the embodiment of the present invention.
  • the present invention provides a preparation method of bone tissue single cell suspension to solve the above problems in the background technology.
  • a preparation method of bone tissue single cell suspension comprising the following steps:
  • the culture medium includes serum-free medium and complete medium, prepare cell cleaning solution, and prepare mixed enzyme dissociation stock solution;
  • Tissue pretreatment transfer the bone tissue and tissue preservation solution to a 15ml test tube, absorb the tissue protection solution; absorb the cell cleaning solution and gently rinse the tissue 1-2 times, and remove the cell washing after standing for 3 minutes each time liquid;
  • step 7) To measure the yield of viable cells, absorb the AO/PI dye solution and the same volume of the target tissue single cell suspension in step 6) to fully mix, then pipette and fill it into the pool in the counting plate, and measure the viable cells by the cell counter. cell ratio.
  • Every 100ml of said serum-free medium contains 99ml of basal medium and 1ml of penicillin-streptomycin mixture, and every 100ml of said medium contains 89ml of basal medium, 10ml of FBS and 1ml of penicillin-streptomycin Mixed solution, the basal medium is ⁇ -MEM medium.
  • the cell cleaning solution is DPBS-5% FBS solution, and the DPBS-5% FBS solution contains 95ml DPBS solution and 5ml FBS solution.
  • the mixed enzyme dissociation stock solution includes lyophilized powder of type II collagenase, lyophilized powder of type I collagenase and serum-free basal medium, and 30 mg of lyophilized powder of type II collagenase is added to every 30 ml of serum-free basal medium and 30mg type I collagenase freeze-dried powder to prepare a mixed enzyme dissociation stock solution, and the serum-free basal medium is ⁇ -MEM medium.
  • the cell filter in step 5) is a 40 ⁇ m cell filter.
  • the method for obtaining the single-cell suspension of the target tissue in the step 6) is as follows:
  • PBS-5% FBS resuspension count 1200rpm, 6min after centrifugation, discard the supernatant; add 250-500 ⁇ l DPBS-5%FBS solution, gently pipette, resuspend and mix; draw 10 ⁇ l resuspended cell suspension and mix with 10 ⁇ l of AO/PI staining solution was mixed and poured into the counting plate, and the original concentration of the cell suspension was obtained after detection by the cell counter.
  • the invention also discloses a bone tissue single cell suspension.
  • the invention also discloses the application of bone tissue single cell suspension in single cell sequencing.
  • Step 1 Configure reagents and media
  • Serum-free medium Aliquoted ⁇ -MEM medium contains 1% Penicillin-Streptomycin; 99ml ⁇ -MEM medium is added with 1ml Penicillin-Streptomycin
  • Step Two Tissue Pretreatment
  • the fourth step the second thorough digestion of the tissue
  • Step 5 Filter the obtained cell suspension with a ⁇ m cell filter
  • Step 6 Obtain single cell suspension of target tissue
  • Step 7 Measure the yield of living cells
  • the results are shown in Table 1 below.
  • the total number of cells is about 3.05*10 5 -1.34*10 6
  • the number of viable cells is between 2.43*10 5 –1.12*10 6 cells/ml
  • the rate of viable cells is between 77.61–84.84%. between.
  • Table 1 is as follows:

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Abstract

提供了一种骨骼组织单细胞悬液的制备方法，包括：1）配制试剂与培养基，2）组织预处理，3）组织初步消化解离，4）组织二次彻底消化，5）μm细胞过滤器过滤，6）获取目的组织单细胞悬液；7）测活细胞得率。该方法能够实现低成本、快速、高效获得骨骼组织单细胞悬液，并保证解离的细胞保持良好活力，可应用于大范围的动物骨骼细胞的解离、分离纯化以及细胞系的建立，为动物单细胞、基因功能及遗传育种研究提供重要参考资料。

Description

一种骨骼组织单细胞悬液的制备方法 技术领域
本发明涉及基因检测技术领域，具体为一种骨骼组织单细胞悬液的制备方法。
背景技术
近年来，随着生物研究领域中技术的飞速发展，单细胞测序技术应运而生，单细胞测序技术是指在单个细胞水平上，对基因组、转录组、表观组进行高通量测序分析的一项新技术。单细胞测序能够揭示单个细胞的基因结构和基因表达情况，对细胞进行分类比较，反映细胞间的异质性，在肿瘤、发育生物学、免疫学、微生物学等领域发挥重要作用。
不同的组织具有各自独特的生理特性，是导致组织细胞培养成为世界性难题的主要原因。现存的实验技术仍不能获得细胞活力较好的组织单细胞悬液，制约着组织的细胞培养、细胞系的建立和功能基因验证等方面研究工作。因此，高效制备组织单细胞悬液有助于建立和优化成体组织细胞的体外培养技术，为细胞系的建立提供重要的参考资料，并在功能基因、遗传育种等研究领域具有重要的理论和实践意义。
发明内容
本发明的提供一种骨骼组织单细胞悬液的制备方法。
本发明的方案是：
一种骨骼组织单细胞悬液的制备方法，包括下列步骤：
1)配制试剂与培养基，所述培养基包括无血清培养基与完全培养基，配制细胞清洗液，配制混合酶解离原液；
2)组织预处理，将骨骼组织与组织保沪液转移至15ml的试管中，吸去组织保护液；吸取细胞清洗液将组织轻轻冲洗1-2次，每次静置3min后去除细胞清洗液；
3)组织初步消化解离，加入所述3-5ml混合酶解离原液；消化孵育，37℃水浴锅，25min；终止消化，加入配制完成的等体积完全培养基溶液，轻轻吸打混匀，肉眼观察不见组织小段，转移至50ml的试管中；
4)组织二次彻底消化，离心去上清液，离心条件1200rpm，5min；重悬孵 育，加入5ml的无血清培养基溶液与5ml的0.25％胰酶细胞消化液溶液，37℃孵育15min，再加入10U/ml脱氧核糖核酸酶I，冷孵育3min；移液枪轻轻吹打，直至组织几乎完全裂解，肉眼观察不见明显团块；终止消化，加入等体积的配制好的完全培养基溶液，轻轻吸打混匀，获得细胞悬液；
5)μm细胞过滤器过滤，将4)中获取的细胞悬液混匀后通过细胞过滤器缓慢吸吹至新的试管中，并在试管管壁上标记；
6)获取目的组织单细胞悬液；
7)测活细胞得率，吸取AO/PI染液与等体积的步骤6)的目的组织单细胞悬液充分混匀后吸打充入计数板中的池中，由细胞计数仪测得活细胞比率。
作为优选的技术方案，每100ml所述无血清培养基中含有99ml的基础培养基与1ml的青链霉素混合液，每100ml所述的培养基中含有89ml的基础培养基、10ml的FBS与1ml的青链霉素混合液，所述基础培养基为ɑ-MEM培养基。
作为优选的技术方案，所述细胞清洗液为DPBS-5％FBS溶液，所述DPBS-5％FBS溶液含有95ml DPBS溶液与5ml FBS溶液。
作为优选的技术方案，所述混合酶解离原液包括Ⅱ型胶原酶的冻干粉、I型胶原酶的冻干粉与无血清基础培养基，每30ml的无血清基础培养基中加入30mgⅡ型胶原酶的冻干粉与30mg I型胶原酶冻干粉，配制混合酶解离原液，所述无血清基础培养基为ɑ-MEM培养基。
作为优选的技术方案，所述步骤5)中细胞过滤器为40μm的细胞过滤器。
作为优选的技术方案，所述步骤6)中获取目的组织单细胞悬液的方法如下：
裂红，经1300rpm,5min离心后弃上清液；加入500μl的红细胞裂解液,重悬细胞混匀,冰上孵育5min，然后加入500μl DPBS。
PBS-5％FBS重悬计数，1200rpm,6min离心后弃上清液；加入250-500μl的DPBS-5％FBS溶液，轻轻吸打,重悬混匀；吸取10μl重悬的细胞悬液与10μl AO/PI染液混匀后打入计数板，细胞计数仪检测后获取细胞悬液原始浓度。
获取目的组织单细胞悬液，用含5％FBS的DPBS细胞清洗液将原始单细胞悬液浓度稀释至8.5*10 5～1.8*10 6cells/ml；
本发明还公开了一种骨骼组织单细胞悬液。
本发明还公开了一种骨骼组织单细胞悬液在单细胞测序中的应用。
由于采用了上述技术方案，一种骨骼组织单细胞悬液的制备方法，1)配制 试剂与培养基，所述培养基包括无血清培养基与完全培养基，配制细胞清洗液，配制混合酶解离原液；2)组织预处理，将骨骼组织与组织保沪液转移至15ml的试管中，吸去组织保护液；吸取细胞清洗液将组织轻轻冲洗1-2次，每次静置3min后去除细胞清洗液；3)组织初步消化解离，加入所述3-5ml混合酶解离原液；消化孵育，37℃水浴锅，25min；终止消化，加入配制完成的等体积完全培养基溶液，轻轻吸打混匀，肉眼观察不见组织小段，转移至50ml的试管中；4)组织二次彻底消化，离心去上清液，离心条件1200rpm，5min；重悬孵育，加入5ml的无血清培养基溶液与5ml的0.25％胰酶细胞消化液溶液，37℃孵育15min，再加入10U/ml脱氧核糖核酸酶I，冷孵育3min；移液枪轻轻吹打，直至组织几乎完全裂解，肉眼观察不见明显团块；终止消化，加入等体积的配制好的完全培养基溶液，轻轻吸打混匀，获得细胞悬液；5)μm细胞过滤器过滤，将4)中获取的细胞悬液混匀后通过细胞过滤器缓慢吸吹至新的试管中，并在试管管壁上标记；6)获取目的组织单细胞悬液；7)测活细胞得率，吸取AO/PI染液与等体积的步骤6)的目的组织单细胞悬液充分混匀后吸打充入计数板中的池中，由细胞计数仪测得活细胞比率。
本发明的优点，本发明制备骨骼组织单细胞悬液的方法，能够实现低成本、快速、高效获得骨骼组织单细胞悬液，并保证解离的细胞保持良好活力，可应用于大范围动物骨骼细胞的解离、分离纯化以及细胞系的建立，为动物单细胞、基因功能及遗传育种研究提供重要参考资料。
附图说明
图1为本发明的制备流程图；
图2为本发明实施例骨骼组织单细胞悬液质检结果(AO/PI)图。
具体实施方式
为了弥补以上不足，本发明提供了一种骨骼组织单细胞悬液的制备方法以解决上述背景技术中的问题。
一种骨骼组织单细胞悬液的制备方法，包括下列步骤：
1)配制试剂与培养基，所述培养基包括无血清培养基与完全培养基，配制细胞清洗液，配制混合酶解离原液；
2)组织预处理，将骨骼组织与组织保沪液转移至15ml的试管中，吸去组织保护液；吸取细胞清洗液将组织轻轻冲洗1-2次，每次静置3min后去除细胞 清洗液；
3)组织初步消化解离，加入所述3-5ml混合酶解离原液；消化孵育，37℃水浴锅，25min；终止消化，加入配制完成的等体积完全培养基溶液，轻轻吸打混匀，肉眼观察不见组织小段，转移至50ml的试管中；
4)组织二次彻底消化，离心去上清液，离心条件1200rpm，5min；重悬孵育，加入5ml的无血清培养基溶液与5ml的0.25％胰酶细胞消化液溶液，37℃孵育15min，再加入10U/ml脱氧核糖核酸酶I，冷孵育3min；移液枪轻轻吹打，直至组织几乎完全裂解，肉眼观察不见明显团块；终止消化，加入等体积的配制好的完全培养基溶液，轻轻吸打混匀，获得细胞悬液；
5)μm细胞过滤器过滤，将4)中获取的细胞悬液混匀后通过细胞过滤器缓慢吸吹至新的试管中，并在试管管壁上标记；
6)获取目的组织单细胞悬液；
7)测活细胞得率，吸取AO/PI染液与等体积的步骤6)的目的组织单细胞悬液充分混匀后吸打充入计数板中的池中，由细胞计数仪测得活细胞比率。
每100ml所述无血清培养基中含有99ml的基础培养基与1ml的青链霉素混合液，每100ml所述的培养基中含有89ml的基础培养基、10ml的FBS与1ml的青链霉素混合液，所述基础培养基为ɑ-MEM培养基。
所述细胞清洗液为DPBS-5％FBS溶液，所述DPBS-5％FBS溶液含有95ml DPBS溶液与5ml FBS溶液。
所述混合酶解离原液包括Ⅱ型胶原酶的冻干粉、I型胶原酶的冻干粉与无血清基础培养基，每30ml的无血清基础培养基中加入30mgⅡ型胶原酶的冻干粉与30mg I型胶原酶冻干粉，配制混合酶解离原液，所述无血清基础培养基为ɑ-MEM培养基。
所述步骤5)中细胞过滤器为40μm的细胞过滤器。
所述步骤6)中获取目的组织单细胞悬液的方法如下：
裂红，经1300rpm,5min离心后弃上清液；加入500μl的红细胞裂解液,重悬细胞混匀,冰上孵育5min，然后加入500μl DPBS。
PBS-5％FBS重悬计数，1200rpm,6min离心后弃上清液；加入250-500μl的DPBS-5％FBS溶液，轻轻吸打,重悬混匀；吸取10μl重悬的细胞悬液与10μl AO/PI染液混匀后打入计数板，细胞计数仪检测后获取细胞悬液原始浓度。
获取目的组织单细胞悬液，用含5％FBS的DPBS细胞清洗液将原始单细胞悬液浓度稀释至8.5*10 5～1.8*10 6cells/ml；
本发明还公开了一种骨骼组织单细胞悬液。
本发明还公开了一种骨骼组织单细胞悬液在单细胞测序中的应用。
为了使本发明实现的技术手段、创作特征、达成目的与功效易于明白了解，下面结合具体实施例，进一步阐述本发明。
实施例1：
第一步：配置试剂和培养基
1.分别准备以下两种培养基
(1)无血清培养基：分装的ɑ-MEM培养基中含有1％的Penicillin-Streptomycin；99mlɑ-MEM培养基加入1ml Penicillin-Streptomycin
(2)完全培养基：分装的ɑ-MEM培养基中含有10％FBS和1％Penicillin-Streptomycin；89mlɑ-MEM培养基加入10ml FBS和1ml Penicillin-Streptomycin
2.准备细胞清洗液：1*DPBS-5％FBS溶液溶液：95ml 1*DPBS溶液与5ml FBS溶液混匀
3.配制混合酶解离原液：将30mg II型胶原酶的冻干粉(Sigma，货号C6885-1G),30mg I型胶原酶的冻干粉(Sigma，货号C0130-1G)溶于10ml的无血清ɑ-MEM培养基(Gibco，货号12571-063)中，待完全溶解后，过滤除菌并保存于-20℃冷冻柜中，避免反复冻融。
第二步：组织预处理
将骨骼组织和组织保沪液转移至15ml的tube管中，吸去组织保护液。吸取细胞清洗液将组织轻轻冲洗1-2次，每次静置3min后去除清洗液。
第三步：组织消化解离
初步消化解离，加入3-5ml混合酶解离原液。消化孵育，37℃水浴锅，25min。终止消化，加入等体积的配置好的完全培养基溶液，轻轻吸打混匀(肉眼观察不见组织小段)，转移至50ml的tube管中。
第四步：组织二次彻底消化
离心去上清液，离心条件：1200rpm，5min。重悬孵育，加入5ml的无血清培养基溶液和5ml的0.25％Trypsin-EDTA溶液，37℃孵育15min，再加入10U/ml DNase I，冷孵育3min。移液枪轻轻吹打，直至组织几乎完全裂解(肉眼观察不见明显团块)。终止消化，加入等体积的配制好的完全培养基溶液，轻轻吸打混匀，获得细胞悬液。
第五步：μm细胞过滤器过滤将获取的细胞悬液
将4)中获取的细胞悬液混匀后通过细胞过滤器缓慢吸吹至新的tube管中，并在管壁上标记。
第六步：获取目的组织单细胞悬液
裂红，经1300rpm,5min离心后弃上清液。加入500μl的红细胞裂解液,重悬细胞混匀,冰上孵育5min，然后加入500μl 1*DPBS。
1*PBS-5％FBS重悬计数，1200rpm,6min离心后弃上清液。加入250-500μl的1*DPBS-5％FBS溶液，轻轻吸打,重悬混匀。吸取10μl重悬的细胞悬液和10μl AO/PI染液混匀后打入计数板，counter star FL20314细胞计数仪检测后获取细胞悬液原始浓度。
获取目的组织单细胞悬液，用含5％FBS的1*DPBS细胞清洗液将原始单细胞悬液浓度稀释至8.5*10 5～1.8*10 6cells/ml；
第七步：测活细胞得率
吸取10μl AO/PI染液和等体积的单细胞悬液，充分混匀后吸取20μl充入计数板中的池中。由counter star的FL20314进行AO-PI质检测得活细胞比率。(图2)
结果如下表1所示，总细胞数在3.05*10 5-1.34*10 6左右，活细胞数在2.43*10 5–1.12*10 6cells/ml之间，活细胞率在77.61–84.84％之间。其中骨N和骨C各三个标本，总共6个标本，均成功进行建库，等待上机测序。
表1如下：
Figure PCTCN2022081254-appb-000001
Figure PCTCN2022081254-appb-000002
以上显示和描述了本发明的基本原理、主要特征及本发明的优点。本行业的技术人员应该了解，本发明不受上述实施例的限制，上述实施例和说明书中描述的只是说明本发明的原理，在不脱离本发明精神和范围的前提下，本发明还会有各种变化和改进，这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等效物界定。

Claims (8)

  1. 一种骨骼组织单细胞悬液的制备方法，其特征在于，包括下列步骤：
    1)配制试剂与培养基，所述培养基包括无血清培养基与完全培养基，配制细胞清洗液，配制混合酶解离原液；
    2)组织预处理，将骨骼组织与组织保沪液转移至15ml的试管中，吸去组织保护液；吸取细胞清洗液将组织轻轻冲洗1-2次，每次静置3min后去除细胞清洗液；
    3)组织初步消化解离，加入所述3-5ml混合酶解离原液；消化孵育，37℃水浴锅，25min；终止消化，加入配制完成的等体积完全培养基溶液，轻轻吸打混匀，肉眼观察不见组织小段，转移至50ml的试管中；
    4)组织二次彻底消化，离心去上清液，离心条件1200rpm，5min；重悬孵育，加入5ml的无血清培养基溶液与5ml的0.25％胰酶细胞消化液溶液，37℃孵育15min，再加入10U/ml脱氧核糖核酸酶I，冷孵育3min；移液枪轻轻吹打，直至组织几乎完全裂解，肉眼观察不见明显团块；终止消化，加入等体积的配制好的完全培养基溶液，轻轻吸打混匀，获得细胞悬液；
    5)μm细胞过滤器过滤，将4)中获取的细胞悬液混匀后通过细胞过滤器缓慢吸吹至新的试管中，并在试管管壁上标记；
    6)获取目的组织单细胞悬液；
    7)测活细胞得率，吸取AO/PI染液与等体积的步骤6)的目的组织单细胞悬液充分混匀后吸打充入计数板中的池中，由细胞计数仪测得活细胞比率。
  2. 如权利要求1所述的一种骨骼组织单细胞悬液的制备方法，其特征在于：每100ml所述无血清培养基中含有99ml的基础培养基与1ml的青链霉素混合液，每100ml所述的培养基中含有89ml的基础培养基、10ml的FBS与1ml的青链霉素混合液，所述基础培养基为ɑ-MEM培养基。
  3. 如权利要求1所述的一种骨骼组织单细胞悬液的制备方法，其特征在于：所述细胞清洗液为DPBS-5％FBS溶液，所述DPBS-5％FBS溶液含有95ml DPBS溶液与5ml FBS溶液。
  4. 如权利要求1所述的一种骨骼组织单细胞悬液的制备方法，其特征在于：所述混合酶解离原液包括Ⅱ型胶原酶的冻干粉、I型胶原酶的冻干粉与无血清基础培养基，每30ml的无血清基础培养基中加入30mgⅡ型胶原酶的冻干粉与 30mg I型胶原酶冻干粉，配制混合酶解离原液，所述无血清基础培养基为ɑ-MEM培养基。
  5. 如权利要求1所述的一种骨骼组织单细胞悬液的制备方法，其特征在于：所述步骤5)中细胞过滤器为40μm的细胞过滤器。
  6. 如权利要求1所述的一种骨骼组织单细胞悬液的制备方法，其特征在于，所述步骤6)中获取目的组织单细胞悬液的方法如下：
    裂红，经1300rpm,5min离心后弃上清液；加入500μl的红细胞裂解液,重悬细胞混匀,冰上孵育5min，然后加入500μl DPBS。
    PBS-5％FBS重悬计数，1200rpm,6min离心后弃上清液；加入250-500μl的DPBS-5％FBS溶液，轻轻吸打,重悬混匀；吸取10μl重悬的细胞悬液与10μl AO/PI染液混匀后打入计数板，细胞计数仪检测后获取细胞悬液原始浓度。
    获取目的组织单细胞悬液，用含5％FBS的DPBS细胞清洗液将原始单细胞悬液浓度稀释至8.5*10 5～1.8*10 6cells/ml；
  7. 如权利权利要求1至6中任一项所述的方法制备的骨骼组织单细胞悬液。
  8. 如权利要求7所述的骨骼组织单细胞悬液在单细胞测序中的应用。
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