A method for simultaneously collecting the inner cell mass and the trophoblast outer layer of a mammalian blastula

Abstract

This invention relates to the field of bioengineering technology, and in particular to a method for simultaneously collecting the inner cell mass and trophectoderm of mammalian blastocysts. The method includes the following steps: (1) after removing the zona pellucida of the blastocyst, the blastocyst is cut into an inner cell mass and a trophectoderm portion, and the cut trophectoderm portion is transferred to a culture medium for collection; (2) the inner cell mass portion obtained in step (1) is sequentially placed in an antibody treatment solution and a complement treatment solution for incubation; (3) the inner cell mass portion treated in step (2) is pipetted to obtain a purified inner cell mass. This method achieves simultaneous and efficient separation of two key cell lines from the blastocyst, fundamentally changing the traditional method's "resource waste" model of only obtaining the inner cell mass while discarding the trophectoderm, and providing more comprehensive experimental materials for embryonic development, placental biology, and stem cell research.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to a method for simultaneously collecting the inner cell mass and trophectoderm of mammalian blastocysts. Background Technology

[0002] Embryonic development is a highly coordinated and dynamic process. In mammals, embryonic development from fertilization to the blastocyst stage can be mainly divided into the following key stages: the union of sperm and egg in the ampulla of the fallopian tube to form a zygote (fertilized egg). Figure 1 After cleavage, the fertilized egg develops into a blastocyst, within which two cell groups form: the trophoblast and the inner cell mass. While the trophoblast does not directly participate in the formation of the embryo itself, it plays a crucial role in processes such as implantation. As the fertilized egg attaches to the uterine lining, the surrounding trophoblast cells rapidly proliferate and differentiate to form the chorionic villi, a key component of the placenta. The placenta originates from the development of the trophoblast, with the proliferation and differentiation of trophoblast cells constituting its core components and performing its primary functions. The subtypes and functions of trophoblast cells are vital in placental biology, and their complex regulatory mechanisms are indispensable for normal placental development. Before successful implantation, the embryo undergoes two main developmental processes: fertilization and the formation of the mammalian blastocyst. The hallmark of blastocyst formation is the establishment of two important cell lines: the trophoblast and the inner cell mass. The trophoblast further forms the blastocyst wall and creates a restricted trophoblast lineage, such as... Figure 1 As shown.

[0003] Subsequently, trophoblast cells proliferate and differentiate into villous or exovulid trophoblast cells, primarily comprising three trophoblast cell subpopulations: cytotrophoblast cells, villous ectodermal trophoblast cells, and syncytial trophoblast cells. Cytotrophoblast cells fuse to form syncytial trophoblast cells. Syncytial trophoblast cells possess two polarized plasma membranes, both expressing nutrient transport proteins (maternally-facing microvilli and basement membranes), and are key regulators of maternal-fetal substrate exchange. Syncytial trophoblast cells transport nutrients and oxygen necessary for growth to the developing embryo through the maternal-fetal circulation, and produce hormones and proteins necessary for maintaining pregnancy. They also act as a barrier against pathogen infection, protecting the fetus from harm.

[0004] Traditional blastocyst isolation methods typically only obtain the inner cell mass, discarding the trophectoderm. As the importance of the trophectoderm in development is increasingly revealed, there is a pressing need for a method that can simultaneously isolate high-purity inner cell mass and trophectoderm. For a long time, in life science research, especially in embryonic developmental biology, cell isolation strategies for blastocysts have often exhibited a rather "utilitarian" selectivity. Traditional methods, like an unwritten rule, usually focus research and prioritize the tiny but precious cell mass inside the blastocyst—the inner cell mass. As the direct source of embryonic stem cells, the inner cell mass is considered the "seed" for the future development of all fetal tissues and organs, and its pluripotency makes it a "star" of research. Therefore, in many experimental designs, isolating and purifying the inner cell mass is the primary and almost sole objective. However, this "emphasis on the inner cell mass, neglect of the trophectoderm" strategy often involves a seemingly "natural" but actually wasteful step—discarding the trophectoderm cells that form the outer shell of the blastocyst, along with the surrounding zona pellucida. As the name suggests, trophoblastic ectoderm cells were initially thought to nourish and protect the inner cell mass, forming extraembryonic structures such as the chorion and placenta. For a long time, they were considered more of a "supportive" or "transitional" structure, and their developmental potential and biological significance were not given sufficient attention.

[0005] As research continues, particularly in areas such as early embryonic development, cell fate determination, epigenetic regulation, and regenerative medicine, the importance of trophectoderm cells is being revealed and reassessed at an unprecedented pace. It is increasingly recognized that the trophectoderm is not merely a "protective shield" for the inner cell mass, but also possesses unique biological functions and value: First, the key drivers of placental formation. The trophoblastic ectoderm is the precursor cell for the formation of a functional placenta. As a crucial organ for the exchange of substances and gases, hormone secretion, and immune barrier between the mother and fetus, the placenta's development directly affects the survival, growth, and even adult health of the embryo. Therefore, in-depth research into the developmental regulatory mechanisms of the trophoblastic ectoderm is essential for understanding pregnancy-related diseases such as preeclampsia and fetal growth restriction.

[0006] Second, it provides a unique cell model. Trophoblast stem cells isolated from trophoblast ectoderm have unique biological characteristics and are an important tool for studying cell lineage determination, cell heterogeneity, stress response, and potential clinical applications (such as as a drug screening model or for the treatment of placental-related diseases).

[0007] Third, it reveals the commonalities and differences in early development. Comparing the developmental pathways, gene expression profiles, and epigenetic modifications of the inner cell mass and trophoblastic ectoderm helps to more comprehensively understand how early embryos differentiate from single cells into cell populations with different fates and functions, which is of fundamental significance for revealing the basic laws of the origin of life.

[0008] Fourth, potential applications in regenerative medicine. Although not as pluripotent as embryonic stem cells, trophoblast-derived cells or their secreted factors may show potential applications in tissue repair and immune regulation. As these important values ​​of the trophoblast are becoming increasingly clear, the traditional practice of discarding it as a waste of valuable biological resources is becoming increasingly inappropriate. Researchers urgently need a smarter, more efficient, and more comprehensive blastocyst processing strategy. The core requirement of this new method is not only "separation" but also "simultaneous separation" and "high purity." "Simultaneous separation" means breaking away from the previous "either / or" choice, allowing both the inner cell mass and the trophoblast to be "captured together," each obtaining independent research samples, thus enabling parallel research or comparative analysis. "High purity" is a stringent requirement for separation quality: if the inner cell mass or trophoblast contains cells from the other or other cell types, it will seriously interfere with subsequent experimental results and affect the accuracy of data interpretation. High-purity separation is the foundation for cutting-edge research such as precise gene editing, epigenetic analysis, and single-cell sequencing.

[0009] Existing methods for isolating the inner cell mass of blastocysts mainly fall into several categories, each with its own operating principle and obvious drawbacks. Tissue culture, by simulating the implantation environment of an embryo in vivo, allows trophectoderm cells to spread and grow in a culture dish, while the inner cell mass cells form a vertically upward columnar structure, which is then picked out using a microscope. Its disadvantages include: low isolation efficiency, requiring a long culture time, and potential contamination if the trophectoderm and inner cell mass are not completely separated; reliance on manual operation, requiring high-precision micromanipulation skills for picking the inner cell mass, placing high demands on the experimentalist's technical level; and decreased cell viability, as prolonged in vitro culture may lead to poor cell condition in the inner cell mass, affecting subsequent experiments.

[0010] Microsurgical methods utilize a micromanipulation system to directly aspirate the inner cell mass cells from the blastocyst for separation. However, they have several drawbacks: high equipment requirements necessitate sophisticated micromanipulation equipment, increasing experimental costs; significant technical difficulty demands highly skilled operators, as even slight errors can lead to damage or loss of the inner cell mass cells; and limited applicability makes them unsuitable for large-scale experiments, hindering widespread adoption.

[0011] Enzymatic digestion utilizes specific enzymes (such as trypsin or collagenase) to break down the junction between the trophoblast and the inner cell mass cells, achieving separation. Its disadvantages include: difficulty in controlling enzyme concentration (too high a concentration may damage the inner cell mass cells, while too low a concentration results in poor separation); impaired cell viability (the enzyme digestion process may cause irreversible damage to the inner cell mass cells, affecting their pluripotency); and limited applicability (currently mainly used in experiments on certain species, such as pigs, and not yet widely adopted).

[0012] In summary, existing methods for separating the inner cell mass each have their advantages and disadvantages, but they generally suffer from the following problems: complex operation, with many methods requiring high-precision equipment or advanced operational skills; high risk of cell damage, as damage to the inner cell mass or trophoblast cells during separation may affect subsequent experimental results; low efficiency, with lengthy separation processes and difficulty in guaranteeing purity; and waste of resources, as trophoblast cells are often discarded, failing to fully utilize their potential value. These shortcomings are precisely the driving force behind the development of a new method capable of simultaneously separating high-purity inner cell masses and trophoblast cells. This new method would not only improve experimental efficiency but also better utilize the potential value of blastocysts, providing broader possibilities for embryonic developmental biology and stem cell research. Summary of the Invention

[0013] The purpose of this invention is to provide a method for simultaneously collecting the inner cell mass and trophectoderm of mammalian blastocysts.

[0014] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for simultaneously collecting the inner cell mass and trophectoderm of mammalian blastocysts, comprising the following steps: (1) After removing the zona pellucida of the blastocyst, the blastocyst is cut into the inner cell mass and the trophectoderm part, and the cut trophectoderm part is transferred to the culture medium for collection; (2) The inner cell mass obtained in step (1) was placed in antibody treatment solution and complement treatment solution for incubation in sequence; (3) The inner cell mass after step (2) is blown to obtain purified inner cell mass.

[0015] Preferably, the cutting in step (1) is performed by drawing a line on the bottom of the droplet in the operating dish with a glass needle, and then fixing the blastocyst with friction.

[0016] Preferably, the cutting needle parameters in step (1) are: HEAT=300, FIL=4, VEL=50, DEL=200, PUL=200.

[0017] Preferably, the antibody treatment solution in step (2) is prepared by mixing guinea pig serum and DMEM culture medium at a volume ratio of 1:0.8~1.2, and the complement treatment solution is prepared by mixing guinea pig total complement and DMEM culture medium at a volume ratio of 1:0.8~1.2.

[0018] Preferably, the antibody treatment solution also contains M2 culture medium and BSA; wherein the amount of M2 culture medium added is 18-22% of the total volume of the antibody treatment solution, and the final concentration of BSA is 0.05-0.15%.

[0019] Preferably, the incubation time of the antibody treatment solution in step (2) is 25-35 min, and the incubation time of the complement treatment solution is 25-35 min.

[0020] Preferably, the blowing in step (3) is performed using a blowing needle prepared by a needle puller and then cauterized to close the opening.

[0021] Preferably, the mammal is a mouse, a human, or a cow.

[0022] The present invention provides a reagent for simultaneously collecting the inner cell mass and trophectoderm of mammalian blastocysts, comprising: an antibody treatment solution prepared by mixing guinea pig serum with DMEM culture medium at a volume ratio of 1:0.8 to 1.2; and a complement treatment solution prepared by mixing total guinea pig complement with DMEM culture medium at a volume ratio of 1:0.8 to 1.2.

[0023] Preferably, the antibody treatment solution also contains M2 culture medium and BSA; wherein the amount of M2 culture medium added is 18-22% of the total volume of the antibody treatment solution, and the final concentration of BSA is 0.05-0.15%.

[0024] In this invention, the specific functions of the antibody treatment solution and the complement treatment solution are as follows: First, the core function of the antibody treatment solution is to "specifically label" trophoblast cells, laying the foundation for subsequent selective clearance. This solution is a mixture of guinea pig serum and DMEM medium at a volume ratio of 1:0.8–1.2, with the addition of M2 medium and BSA to maintain embryo viability. Guinea pig serum contains specific antibodies against surface antigens of trophoblast cells. During incubation, the Fab fragment of the antibody specifically binds to the antigens on the trophoblast cell membrane, forming an antigen-antibody complex, while the Fc fragment of the antibody is exposed, becoming a "tag" for the complement system. This stage only completes the immunolabeling; cell morphology does not change significantly, thus avoiding non-specific damage.

[0025] Second, the complement treatment solution acts as a "killer," specifically lysing labeled trophoblast cells via the classical complement pathway. This solution is a mixture of guinea pig total complement and DMEM culture medium at a volume ratio of 1:0.8–1.2. When the embryo is transferred to the complement treatment solution, the C1q component of the complement recognizes the antibody Fc fragment, sequentially activating factors C4, C2, and C3, ultimately assembling on the target cell membrane to form a membrane attack complex. This leads to changes in trophoblast cell membrane permeability, cell swelling and shrinkage, darkening of color, and gradual detachment. This process is highly dependent on the preceding antibody labeling step, thus having minimal impact on inner cell mass cells, effectively achieving the separation of the trophoblast from the inner cell mass.

[0026] Third, the two treatment solutions work synergistically in stages, ensuring the specificity of the separation while reducing the risk of damage to the inner cell mass. Separating antibody incubation and complement killing into two independent steps allows for precise control of the reaction time and conditions at each step, preventing non-specific activation of complement through alternative pathways when sufficient antibody guidance is lacking. The addition of BSA to the antibody treatment solution helps maintain embryonic osmotic pressure stability, while M2 medium provides necessary nutritional support; the complement treatment solution is diluted 1:1, preserving sufficient complement activity while reducing the cytotoxicity that high-concentration complement may cause. This improved protocol efficiently removes trophoblastic ectoderm residues while maximizing the preservation of the integrity and activity of the inner cell mass, providing a crucial guarantee for obtaining high-purity inner cell masses subsequently.

[0027] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a method for simultaneously collecting the inner cell mass and trophoblast of mammalian blastocysts, the beneficial effects of which are mainly reflected in the following four aspects: First, this method achieves simultaneous and efficient separation of two key cell lines from the blastocyst, fundamentally changing the traditional method's "resource waste" model of only obtaining the inner cell mass while discarding the trophectoderm. By first mechanically cutting and separating the trophectoderm and collecting it separately, and then performing immunological purification on the remaining inner cell mass, it ensures that both cell types can be obtained as independent and complete research samples, providing more comprehensive experimental materials for embryonic development, placental biology, and stem cell research.

[0028] Secondly, this method significantly lowers the operational threshold and improves separation efficiency and throughput. The entire operation can be completed under a regular stereomicroscope, eliminating the need for expensive micromanipulation systems. The cutting step involves fixing the blastocyst by scribing with a glass needle, making the operation simple and highly controllable. Experimental data show that the separation efficiency of this method can reach approximately 100%, and it is applicable to various mammals such as mice, humans, and cattle, meeting the needs of large-scale sample processing.

[0029] Third, this method effectively improves the purity of the inner cell mass through a modified complement-mediated immunopurification step. Based on mechanical cutting, diluted guinea pig serum and total guinea pig complement are used as antibody treatment solutions, and M2 medium and BSA are added to optimize the reaction system. This specifically removes residual trophoblast cells adhering to the surface of the inner cell mass. After pipetting, high-purity inner cell masses with clear morphology and compact structure are obtained, providing high-quality samples for subsequent precise analyses such as gene editing and single-cell sequencing.

[0030] Fourth, this method maintains cell viability while ensuring the complete collection and subsequent utilization value of the trophectoderm. Since all cells are used for sequencing, it avoids changes in cell state caused by in vitro culture, making it particularly suitable for molecular biology research that requires a high degree of attention to the original state of cells. This method not only improves the utilization efficiency of blastocyst resources but also provides crucial technical support for in-depth exploration of the function of the trophectoderm in placental formation, maternal-fetal interaction, and disease model construction. Attached Figure Description

[0031] Figure 1 This refers to the process of mammalian embryonic development and embryo implantation.

[0032] Figure 2 An embryo that has developed a blastocoel.

[0033] Figure 3 For blastocyst lineage differentiation - ICM and TE.

[0034] Figure 4 The blastocyst after separation.

[0035] Figure 5 The ICM obtained by separation.

[0036] Figure 6 This is an RNA-seq heatmap showing the expression of ICM and TE-specific genes in different samples (Z-score normalized). The four sections of the left-hand band, from top to bottom, represent Early ICM, Late ICM, Early TE, and Late TE, respectively.

[0037] The Early ICM includes (listed in order of heatmap bands, the same below): Cpn1, Cst7, Asns, Pgam1, Wdr55, Rps27, Fkbp11, Tmem107, Maged1, Zc3hc1, 4930579G24Rik, Helb,Dram1, Phlda2, Fam178b, Mcf2, Stmn1, Enpp3, Pih1d1, Sparc, Ezr, Ppp1r14d,2610318N02Rik, Zscan10, Uap1, Msh2, Cdca7, Rnf130, Tmem39a, Fcgr3, S100a11, Cdx2, Pla2g15, Tmem62, Epcam, Pkp2, Stx3, Stard10, Gls, Btg4, Cnn2. Sh3bgrl2,Aqp9, Cpne8, Csf3r, Mgst3, Vamp8, Tspan8, Lrrfip1, Hsd17b6, Smpdl3a, Tcfl5,Dkkl1, Oosp1, Clic4, Myl12b, Abracl, Tdpoz2, Cmbl, Trappc3, Car2, Mogat2,Serpinh1, Cryga, Crygc, Slc6a15, Hspb8, Chac1, Gfpt2, Nostrin, Dkk1, Casq2,Capsl, Bex2, Gm19510, Nodal, Sapcd1, Rnase12, Amacr, Creb3l2, Spp1, Smtnl2,Nat1, Clec4a1, Slc13a5, Tinagl1, S100a16, Wnk2, Cited2, Sh2d4a, Mbd2, Pisd-ps1, Entpd1, Rnf208, E2f8, Tacstd2, Gng2, Anxa3, Capn2, Cdk14, Ndrg1, Cmtm8,Tspan7, Txndc16, Atp12a, Acot2, Gulp1, Enpp1, Pdzk1ip1, Smyd1, Rbms1,Pip5k1b, Sh3bp5, Slc22a4, Cln6, Lurap1l, B3gnt5, Ppp2r5a, Mbnl2, Mbnl3,Acsl6, Ldoc1, C430049B03Rik, Il6, Tead2, Prss8, Efhd2, Tmprss2,Dmkn,3830417A13Rik; Late ICM includes: Snx10, Apoe, Apom, Utf1, Cryge, Crygs, Zfp959, Mboat2, Bcas3, Rnd3, Nanog, Hspb1, BC028528, Tet1, Pcolce2, Nup62cl, 4930444P10Rik, Gm13128, Rhebl1, Pdlim4, Tmem41a, Rgs19, Amn, Atxn3, Fbp2, EU599041, Ahcy, Spry2, Itpk1, Etv5, Lgals9, Eif4ebp1, Nupr1, Pfkp, Apobec3, Pim2, Gata4, Gm13212, Trit1, Chek2, Gm11517, Trp53, Stmn3, Tat, Tgoln2, Poglut1, Akr1c21, Icam1, 2810429I04Rik, Snai1, Pnliprp2, Stk31, Plod1, Sh3bp4, Ifitm2, Obox6, Lrrc2, Zfp819, Gfra3, Zfp296, B3gnt7, Spic, Ralb, Tppp3, Parp9, Ube2l6, Gja1, Tuba3a, Lck, Pecam1, Ckb, Psat1, Timp1, Gas5, Tdgf1, Gpx2, Car4, Slc6a8, Ybx2, Fam107b, Btbd1, AU021092, Tmem79, Snx1, Gm12169, Neu1, Tmsb4x, Fbxw14, Fbxw28, AA467197, Gsto1, Slc44a4, Camk1, Msn, Slc34a2, Ovol1, Gyg, Chka, B2m, Egln1, Mmgt2, Mon1b, Vill, Il17re, Abca3, Folr1, Oog3, Pank3, Flot2, Accsl, E330034G19Rik, E330021D16Rik, S1pr5, C86187, Fgfr2, Spsb4, Fbxl12, Cebpa, Smpd1, Nlrp14, Lpcat4, Obox1, Obox5, Gm2a, Neat1, Cish, Dcxr, 2610528J11Rik, Bcl2l10, Lrrn4, Fam151a, Vasp, Sgpp1Rpia, Ankrd13a, Ppp1r3b, Trpv5, Omt2b, Atp6v0a4, Echdc3, Tcl1b1, D6Ertd527e, Plac1, Npepl1, Efnb2, D330041H03Rik, Slc39a2, Clec2i, Cnksr3, Bmp4, Crygd, Mmp1b, Nefl, Hmga1, Lrrc34, Zic3,Prss35, Slc1a4, Aqp8, Tnfrsf8, Triml1, Raet1e, Sfrp1, Ankrd45, Akr1c13,Icam2, Eras, Trh, Aoah, Car14, Pga5, Ldha, Gsdmd, Mid1ip1, Cryab, Cited4, Ahnak, Vgll1, Ccdc69, Hspb2, Fmnl2, Ptms, Mbp, Dsp, Serpinb6b, Scin, B4galt5, A930005H10Rik, Nudt7, Abcc4, Gata2, Eomes, Sema3e, S1pr1, Tmem213, Gm5622, Wnt3a, Gm4926, Hs3st3b1, Cib2, Cfc1, Samd10, Rab15, Cd82, Abhd14b, 3830403N18Rik, Gab2, Ptprf, Pnma5, Tcl1b2, Tcl1b3, Slc40a1, Emp2;, Early TE includes: Apex1, Fcgr2b, Mboat1, Hormad1, Magea2, Angptl4, Rangrf, Xrcc5, Fabp5, 1500009L16Rik, Foxh1, Dnajc22, Hand1, Tsix, Parp1, Morc1, Ifi35, Wdr26, Actn4, Gm10436, Tmem109, Fbxw24, Gm9, Oxct1, Atp6ap1l, Lgals1, Trmt2b, Cacnb3, Fxyd4, Bmp15, Csrp1, Errfi1, Psap, Ptges, Cbs, Ube2r2, Dab2, Cyp2b23, Dcaf12l1, Plcxd3, Dhrs4, Dse, Serpinb1a, F3, Slc1a3, Slc7a3, Efcab10, Sccpdh, Sox2, St3gal4, Gm5662, Eci3, Gm2016, Plod2, Slc25a1, F2r, Upp1, Manba, Lmo2, Plscr1, Ephx2, Fn1, 4930452B06Rik, Tmeff1, Xkrx, Slc6a14, Ubqln2, Tmem171, Nlrx1, Glipr1, Lgals4, Ggta1, Nxf7, Enpep, Klk7, Wls, Slc22a5, Adh1, Ryk; Late TE packages: Ly6g6e, Ostm1, Amhr2, Serping1, Guca1a, Pgam2, Fam168a, P4ha1, Phf6, Rpp25, Dhrs7, Slc5a4b, Stfa1, Mep1b, Isyna1, Mif, Mtmr7, Cd24a, Gm5547, Pkm, Plbd1, Casp3, Pyy, Zfp1, Cbr1, Fez1, Bhmt, Zfp105, Fscn1, Mfge8, Tcf23, Hesx1, Pramef17, Mgarp, Tada2a, Rbpms2, Baiap2, Prdm14, Pycr2, Tex19.2, Gcat, Tdh, Tmem253, Pold2, Porcn, Klb, Efhb, Gulo Ramp2, Thop1,Ly6e, Ifitm3, Smpdl3b, 2200002J24Rik, Ppm1a, Arl6, Zfp534, Inpp5d, Epha4,Qdpr, Pmm1, Pdpn, Elovl5, Pramef8, Tmem106a, Popdc3, Nkx6-2, Jam2, Id3, Gdf3,Gpc4, Fgf4, Gamt, Efhd1, Cth, Ggt1, Cdc42ep3, Txnip, Aktip, Zfp947, Bmyc,Cldn4, Perp, BC053393, Myh10, BC051665, Gsta1, Tmem125, Hsbp1l1, Apoa1,Fabp3, Anxa2, Anxa9, Otud1 Rragd, 1810019D21Rik, Chmp4b, Kank1, AU022751,Gata3, Pon2, St3gal6, Rfpl4, Khdc1a, Khdc1b, Arpc3, Hk2, Krt8, Rab21, Oas1d,Rac1, Fam122b, Sidt2, Crip1, Akr1b8, Tpm1, Naga, Gm13023, Aamdc, Ldhb,Gm10639, Krt18, Efnb1, Gnai2, Gm3776, Lars2, Dpp4, Col4a1, Wdr31, Il6st,Fam25c, Trim44, Ckap4, Nnmt, Mal, Otx2, Fgf10, Them5Fabp9, Pmaip1, Vegfc,Srgn, Rcn1, 2410137M14Rik, Abcg1, Flrt3, Aldh3a1, H2-M5, Lefty2, Emb,Slc22a16, H2-Eb1, 1700019D03Rik, Cd38, Ube2dnl1, Qpct, Cpxm1, Syne4, Myod1,Mgmt, Psmb9, Prl8a9, Lgr4, Gjb4, Ubd, Cd109, Stambpl1, Nt5e, Cpe, Slc46a3,Dync2li1, Vgll3, Limd1, Snx7, Acer1, Dppa1, Gstk1, Ank, Prss32, Gpr85,Mapre3, Oog4, Aldh3b2, C87499, Lrp2, Jund, Fxyd6, Gm1965, Slc15a2, Oas1f,Trpm6, 1700001L19Rik, Pgm2, Gm13084, Vtcn1, Gm10046, Id2, Cers3, Epn3. , Detailed Implementation

[0038] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0039] Example 1

[0040] 1. Blastocyst culture

[0041] Embryos were rinsed out under sterile conditions with PBS containing 5% fetal bovine serum (FBS) Gibco (Life Technologies, USA) catalog number: 16000-044, and then washed three times with M2 medium before use.

[0042] After obtaining a preliminary batch of mouse blastocysts, due to potential inconsistencies in developmental progress, they were all cultured in KSOM medium until larger cavities appeared before further processing. Figure 2 At this stage, the embryos are divided into two lineages: ICM and TE. Figure 3 ).

[0043] 2. Remove the transparent band.

[0044] Remove the zona pellucida using acidic Tyrode's solution (T1788; Sigma-Aldrich; St.).

[0045] 3. Cutting, parameters of the cutting needle

[0046] Instrument: Sutter Instrument Co., Parameters: HEAT=300, FIL=4, VEL=50, DEL=200, PUL=200

[0047] First, draw a line on the bottom of the droplet in the petri dish using a glass needle. This friction helps the blastocyst adhere to the drawn area, preventing it from rolling during cutting and hindering the process. Rotate the blastocyst so that the ICM region is on one side, at the six o'clock position, for easier manipulation. Under a dissecting microscope, use an oral pipette and a needle (manual pulling of the needle is sufficient) to separate the trophectoderm (TE) and inner cell mass (ICM) (which requires further purification). Figure 4 )

[0048] 4. TE transfer

[0049] The embryo transfer needle aspirates TE, washes, and transfers to the culture medium (KSOM(MR-106-D; Millipore); Embryoculture medium).

[0050] 5. ICM separated using a modified complement method

[0051] The inner cell mass fractions were placed sequentially in the antibody treatment solution and incubated for 30 minutes. The antibody treatment solution was prepared by mixing guinea pig serum (anti-rat serum Sigma M5774) and DMEM medium at a volume ratio of 1:1. M2 medium and BSA were also added to the antibody treatment solution. The amount of M2 medium added was 20% of the total volume of the antibody treatment solution, and the final concentration of BSA was 0.1%.

[0052] 4. Transfer the inner cell mass into M2 reagent and wash the blastocyst three times by rolling. 5. Transfer the inner cell mass into the complement treatment solution and incubate for 30 minutes. Changes will be observed in the embryo at this time, including darkening of color and shrinkage (these changes are affected by temperature and embryonic condition; if no obvious changes are observed, continue incubation, observing every 10 minutes, and do not over-incubate). The complement treatment solution is prepared by mixing guinea pig total complement (Guinea Pig Total Complement (GPS) Millipore Cat 234395) with DMEM medium at a 1:1 volume ratio.

[0053] 6. After digestion, the inner cell mass is blown apart. It is recommended to use a syringe pipette to make a blow-apart needle and then cauterize the opening to seal it.

[0054] 7. Wash the ICM clumps with M2 reagent and set aside.

[0055] The isolated ICM cells, when observed under a microscope, resemble pearls—transparent, lustrous, and with compact cell clusters. Figure 5 .

[0056] Experimental Example 1

[0057] Four batches of experiments (all mice) were conducted according to the method in Example 1, and the statistical results are shown in Table 1. Figure 4 and Figure 5 As can be seen, the cut ICM and TE fragments were morphologically intact. The applicant further sent them to a bioassay company for RNA-seq, and the results showed that they each expressed their own marker genes, indicating very pure lineage separation. Figure 6 As can be seen, ICM-specific genes are significantly highly expressed in E3ICM samples (red) and lowly expressed in E4STE samples (blue); while TE-specific genes are highly expressed in E4STE samples and lowly expressed in E3ICM samples. This strong expression specificity indicates that the expression patterns of their respective specific genes are highly differentiated in the ICM and TE cell populations, suggesting that the ICM and TE cell populations are relatively pure.

[0058] Table 1 Statistical Results

[0059] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for simultaneously collecting the inner cell mass and trophectoderm of mammalian blastocysts, characterized in that, Includes the following steps: (1) After removing the zona pellucida of the blastocyst, the blastocyst is cut into the inner cell mass and the trophectoderm part, and the cut trophectoderm part is transferred to the culture medium for collection; (2) The inner cell mass obtained in step (1) was placed in antibody treatment solution and complement treatment solution for incubation in sequence; (3) The inner cell mass after step (2) is blown to obtain purified inner cell mass.

2. The method according to claim 1, characterized in that, The cutting in step (1) is performed by drawing a line on the bottom of the droplet in the operating dish with a glass needle and fixing the blastocyst with friction.

3. The method according to claim 1, characterized in that, The cutting needle parameters in step (1) are: HEAT=300, FIL=4, VEL=50, DEL=200, PUL=200.

4. The method according to claim 1, characterized in that, The antibody treatment solution in step (2) is prepared by mixing guinea pig serum and DMEM culture medium at a volume ratio of 1:0.8~1.2, and the complement treatment solution is prepared by mixing guinea pig total complement and DMEM culture medium at a volume ratio of 1:0.8~1.

2.

5. The method according to claim 4, characterized in that, The antibody treatment solution also contains M2 culture medium and BSA; wherein the amount of M2 culture medium added is 18-22% of the total volume of the antibody treatment solution, and the final concentration of BSA is 0.05-0.15%.

6. The method according to claim 1, characterized in that, The incubation time of the antibody treatment solution in step (2) is 25-35 min, and the incubation time of the complement treatment solution is 25-35 min.

7. The method according to claim 1, characterized in that, The blowing in step (3) is performed using a blowing needle prepared by a needle puller and then cauterized to close the opening.

8. The method according to claim 1, characterized in that, The mammals mentioned are mice, humans, or cattle.

9. A reagent for simultaneously collecting the inner cell mass and trophectoderm of mammalian blastocysts, characterized in that, include: The antibody treatment solution was prepared by mixing guinea pig serum with DMEM culture medium at a volume ratio of 1:0.8~1.2; and the complement treatment solution was prepared by mixing total guinea pig complement with DMEM culture medium at a volume ratio of 1:0.8~1.

2.

10. The reagent according to claim 9, characterized in that, The antibody treatment solution also contains M2 culture medium and BSA; wherein the amount of M2 culture medium added is 18-22% of the total volume of the antibody treatment solution, and the final concentration of BSA is 0.05-0.15%.

Images