KR20020018143A - Human embryonic stem cells derived from frozen-thawed embryo - Google Patents

Abstract

PURPOSE: Provided is a method for culturing and growing undifferentiated human embryo stem cells derived from frozen-thawed embryo. Also, provided is a method for differentiating the undifferentiated stem cells into a specific cell. CONSTITUTION: Undifferentiated human embryo stem cells are prepared by thawing a human embryo in blastocyst stage, and culturing a part of or a whole embryo above on a medium which keeps undifferentiated embryo stem cells. Where, the human embryo in blastocyst stage is frozen 5-6 days after fertilization for over 4 years; and a used cryoprotectant can be selected from the group consisting of sucrose, glycerol and their combination.

Description

Human Embryonic Stem Cells Derived From Frozen-Thawed Embryo}

The present invention relates generally to undifferentiated human embryonic liver cells (ES) derived from freeze-thawed human embryos and to methods for culturing and propagating the undifferentiated embryonic liver cells. The invention also relates to a method of differentiating certain cells from undifferentiated human embryonic liver cells derived from freeze-thawed human embryos.

In fact, hepatocytes are undifferentiated cells that can differentiate into mature cells of various functions ranging from neurons to muscle cells. Embryonic liver cells (hereinafter referred to as "ES" cells) are derived from embryos and are in fact pluripotent. ES cells can differentiate into specific cells, tissues, and even organ types, depending on the type of stimulus they receive.

The development of mouse ES cells was described in 1981 by Evans et al., Nature 292: 151-156; Martin, Proc. Natl. Acad. Sci. USA 78: 7634-7638, and was associated with a study of mouse malformation cancer. Teratogenic cancer research developed extensively in the 1970s and led to the study of EC cells as a model for mammalian cell, tissue and organ differentiation studies in relation to the background developmental capacity of EC (embryonic carcinoma) liver cells. However, EC cells are neoplastic and they necessarily contain chromosomal abnormalities that limit their ability to differentiate into various tissue types.

Malformation cancer has been hypothesized to be inductively induced into blastocyst grafts and that pluripotent cell lines can arise directly from blastocysts instead of tumors. The generation of mouse ES cells was reported in 1981 by Martin and Evans et al. This result was a stable diploid cell line that is said to be able to generate all adult tissue types. Malformations have also been reported to arise from primordial germ cells and translocation of primordial germ cells in mice. In 1992, Matsui et al. Reported the acquisition of EG cells (embryonic germ cells) from mouse primordial germ cells ( Cell 70: 841-847). EG cells have a developmental capacity very similar to ES cells. Because mouse ES cells can differentiate into any humoral cell type, mouse ES cells can be used in vitro to study the mechanisms that regulate the differentiation of specific cells or tissues. While studies on mouse ES cells provide an understanding of the general differentiation of mammalian cells and tissues, the use of mouse ES cells as a human developmental research model has been limited because of the development of primates and mice and differences in specific cell lines.

In addition, pluripotent cell lines were derived from testicular cancer (Andrews et al., 1984, Lab. Invest. 50: 147-162), and induction of cloned cell lines from human malformations that can differentiate into neurons and other cell types in vitro. Was reported. In addition, cell lines capable of differentiating into representative tissues of all three types of embryonic germ cell layers were generated (Pera et al., 1988, Differentiation 39: 139-149). These studies on human cell development show that these derivatives are aneuploid, have limited capacity for spontaneous differentiation into somatic tissue, and differ from the phenotype of mouse ES or EC cells.

The development of primate ES cells from blastocysts of rhesus monkeys and pythons has been published (Thomson et al., 1995, Proc. Natl. Acad. Sci. USA 92: 7844-7844, 1996, Biol. Reprod. 55: 254 -259). Although these primate cell lines are diploids, they are very similar to human EC cells in other respects. These studies on monkey cells show that studies of primates, including human EC cells, differ in mouse phenotype and are related to pluripotent liver cells that can be derived from human blastocysts.

Bonso et al . Reported the short-term culture and maintenance of cells from fertilized human embryos in vitro ( Human Reprod. 1994, 9: 2110-2117). The isolated cells had the form expected from pluripotent liver cells, but these early studies did not use feeder cell scaffolds and also made it impossible to maintain the culture for a long time. Thompson et al. (1998, Science 282: 1145-1147) induced ES cells from human blastocysts by immunoresection to remove ectoderm, followed by laying internal cell masses on the mouse embryonic fibroblast feeder cell layer. Attached and expanded, and the extroverts formed were decomposed and laid back on other feeder cell layers. The phenotype of the resulting cells is similar to human EC cells reported by Pera et al.

Thompson's work on primate ES cells showed no evidence that these cells had somatic differentiation in vitro. The only evidence for in vitro differentiation is limited expression of specific markers of trophoblast and endoderm formation, such as the production of human chorionic gonadotropin alpha fetoprotein. It is difficult to demonstrate that the cells producing alpha-fetoprotein are equivalent to constituting extra embryos or embryonic endoderm.

Methods for establishing ES cells by culturing undifferentiated primordial germ cells from 5 to 9 week old aborted fetuses are described in the literature (Shamblott et al., 1998, Proc. Natl. Acad. Sci. USA 95: 13726). However, this method does not provide a method for differentiating ES cells from high quality ES cells or ES cells derived from frozen embryos into specific cells.

Rubinoff et al. Published a paper on ES cells derived from relatively neoblastocyst embryos six days after fertilization (2000, Nature 18: 399-404). Rubinov reports on the proliferation of undifferentiated human ES cells and the production of human ES cells capable of producing differentiated somatic cells. However, this method did not provide an effective way to produce high quality ES cells from frozen embryos, which have been long term preserved.

Human EC cells will form teratocarcinomas with derivatives of multiple embryonic tumor strains in nude mice. However, the differentiation range of these human EC cells is limited compared to the differentiation range obtained with mouse ES cells, and all induced EC cell lines are diploids (Andrew et al., 1987, supra). On the other hand, ES cells have greater development potential because they are derived from normal embryonic cells in vitro without the pressure of choice for the teratocarcinoma environment. True ES cells must (i) be able to multiply in vitro in an undifferentiated state, (ii) maintain a normal karyotype through prolonged culture, and (iii) all three germ germ layers (endoderm) after prolonged culture. , Mesoderm and ectoderm), the potential to differentiate into derivatives.

Since human ES cell lines will facilitate the study of early human development, any method that allows for the production of human ES cells is desirable, and also using such human ES cell lines for implantation, manufacture of biopharmaceutical products, and development of detectors based on biology It will be possible to develop a cell culture method for. Importantly, the ability to produce large amounts of human cells can be achieved by the preparation of substances such as insulin or VIII factors that must be obtained from sources or donors that are not currently human; Transplantation to treat diseases such as Parkinson's disease; Tissue for use as a graft; And important practical applications for the screening of drugs and toxins.

An object of the present invention is to solve the problems according to the prior art, as described above, after freezing and storing the human blastocyst embryo 5-6 days old after fertilization, liver cells from human embryos to be discarded after a certain time To provide an effective way to establish.

1 is a micrograph (X200) of a human blastocyst embryo frozen after thawing,

2 is a micrograph (X200) of the frozen-thawed human blastocyst embryo in the expanded state after culture;

3 is a micrograph (X200) of a human blastocyst embryo from which a zona pellucida was removed by pronase treatment

Figure 4 is a micrograph (X200) of the human blastocyst embryo in the state of trophectoderm necrosis by immunotomy;

5 and 6 are micrographs (X200) of internal cell mass (ICM) from residual blastocyst embryos after complete removal of trophectoderm;

FIG. 7 is a micrograph (X400) of placing ICM cells on STO cells; FIG.

8 is a micrograph (X300) of ICM colonies formed 7 days after initial plating,

9 is a micrograph (X80) of an ICM colony formed after 13 days of replating the ICM colony of FIG. 8,

10 is a micrograph (X150) of mechanically isolated and cultured ICM cells,

11 is a micrograph (X200) of one colony of human ES cells cultured for 5 weeks,

12 is a micrograph (X400) magnifying the colony of FIG. 11;

Figure 13 is a micrograph (X 80) of the differentiated cells that are not stained as a result of alkaline phosphatase activity measurement,

14 is a micrograph (X300) of human ES cells stained as a result of alkaline phosphatase activity measurement,

15 is a micrograph (X40) of embryonic liver cell colonies differentiated into cardiomyocytes,

16 is a micrograph (X60) showing the formation of cardiomyocytes according to the method of the present invention,

17 is a micrograph (X100) of cardiomyocytes differentiated and beating according to the method of the present invention,

18 is a micrograph (X100) of a typical neuron,

19 is a micrograph (X40) of neurons stained with 200 kDa anti-nerve microfibrillary protein,

20 is a micrograph (X600) of neurons stained with anti-microtubule binding protein 2,

21 is a micrograph (X600) of neurons stained with anti-β tubulin,

22 is a micrograph (X100) of a typical glial cell,

FIG. 23 is a micrograph (X100) of glial cells stained with anti-glial fiber protein.

FIG. 24 is a micrograph (X200) of glial cells stained with anti-galactocelebrocide,

25 and 26 show micrographs (X400) of muscle cells stained with anti-muscle actin,

Figure 27 shows the results of electrophoresis to compare the degree of Oct-4 expression in the embryonic body (EB) and ES cell colonies,

28 shows the results of electrophoresis to compare expression levels of Oct-4b in colonies of ES cells and differentiated ES cells,

29 is a micrograph (X60) of freeze-thawed human ES cells prepared according to the method of the present invention,

30 is a medium spread,

31 karyotype of human ES cell line.

The present invention relates to methods of establishing undifferentiated human embryonic liver (hereinafter referred to as "ES") cells and to undifferentiated human ES cell lines formed therefrom.

In one aspect of the invention, there is provided a method of establishing undifferentiated human ES cells, the method comprising the steps of fusing cryopreserved human embryos, and removing some or all of the embryos on a medium capable of maintaining undifferentiated embryonic liver cells. Culturing.

In another aspect of the invention, the ES cells are from freeze thawed human blastocyst embryos. The method includes fusion of cryopreserved human blastocyst embryos and culturing some or all of the embryos on a medium capable of maintaining undifferentiated embryonic liver cells.

In another embodiment of the present invention, there is provided a method of establishing undifferentiated human ES cells, which comprises obtaining a population of cryopreserved human embryos in developing blastocysts, fusing one or more embryos and undifferentiating Culturing some or all of each embryo fused on a medium capable of maintaining the ES cells.

In another aspect of the present invention, a method for establishing undifferentiated human ES cells comprises the steps of fusing cryopreserved human embryos in a solution comprising human follicular fluid (hereinafter referred to as "hFF") and hfused and frozen thawed human embryos. It is characterized by a method comprising a continuous step of treating in two or more solutions containing an inhibitor.

In another aspect of the invention, the method further comprises the step of removing trophectoderm from the embryo using an anti human lymphocyte antibody. In a preferred method of establishing undifferentiated human ES cells, the embryo part comprises an internal cell mass (hereinafter referred to as "ICM").

The present invention also relates to a method for separating ICM from human blastocyst embryos comprising treating the embryo with an anti human lymphocyte antibody. Preferably, the method of isolating ICM further comprises human blastocyst embryos, which are cryopreserved and thawed.

In another aspect of the present invention, a method of differentiating human ES cells is provided, the method comprising the step of differentiating ES cells obtained in the method of the present invention in a culture medium containing a growth factor.

These features will be described in conjunction with the accompanying drawings.

The present invention provides methods for establishing undifferentiated human embryonic liver (ES) cells from freeze-thawed embryos and undifferentiated human ES cell lines formed from such cells. In particular, the present invention provides a method for establishing undifferentiated human ES cells from freeze-thawed human blastocyst embryos.

The invention further relates to a method of differentiating human ES cells into specific cells.

The development of ES cell cultures that can be maintained as cell lines allows the study of fundamental questions about the biochemical and cellular properties of these cells and their dynamic interactions in their cellular and chemical environments. In addition, the human ES cells of the present invention can be used to produce useful tissues and materials.

An important feature of the present invention is the use of freeze thawed embryos. Most prior art used fresh fertilized eggs obtained from in vitro fertilization (IVF). In fertility clinics, in general, it is difficult to obtain consent from parents on the use of such fertilized eggs for research purposes, since the fertilized eggs are frozen and later frozen. However, a feature of the present invention is the use of fertilized eggs that are frozen for a period of time, preferably not implanted and then discarded after several years of freezing. Thus, because the present invention uses frozen fertilized eggs that are no longer needed or will be discarded, the present invention makes it possible to use large numbers of fertilized eggs for ES cell production. In addition, frozen embryos may be used with parental consent, even if not discarded. In addition, since the use of unnecessary or discarded fertilized eggs can be used, it can be used more freely without great difficulty in avoiding ethical problems. Therefore, the most advantageous feature of the present invention is the successful induction of ES cells from frozen embryos, especially those which are not needed or to be discarded, which have been long term preserved.

Another feature of the invention is the use of frozen blastocyst embryos. A large number of fertilized eggs (ie embryos) die in the early stages of embryonic development, especially in the embryonic stage within about 2 to 3 days after fertilization. These embryonic development periods in particular generally involve both electrical and lossy exhaustion. Thus, the number of surviving embryos is very limited when using 2-3 day old embryos. On the other hand, blastocysts are more viable. Thus, the possibility of establishing ES cells from frozen embryos cultured up to the blastocyst is a possibility of using 2-3 days old embryos not cultured up to blastocyst embryos, such as those described in Thomson's US Pat. No. 6200806, incorporated herein by reference. Much higher than that. By doing so, it is possible to considerably save time and costs for establishing ES cells.

A further feature of the present invention is the use of anti human lymphocyte antibodies in the immunoresection of embryos. The inventors of the present invention have found that anti human lymphocyte antibodies are more effective at removing trophectoderm from blastocyst embryos than other antibodies commonly used such as anti human serum antibodies.

Justice

The following terms are defined as set forth unless otherwise stated. All other terms used herein are defined with respect to the meanings used in the specific field concerned, unless otherwise indicated.

The term “embryo”, as defined herein, refers to an embryo up to the start of 3 months post-pregnancy, in which organs and organ systems begin to develop after fertilization.

The term "cultured embryo" refers to an embryo before implantation consisting of a ball or sphere of cells with an outer cell layer, a liquid packed cavity and an inner cell mass. As will be appreciated by those skilled in the art, such spheres need not be ideal geometric spheres, but instead should be spherical in form of cells. Generally, blastocysts will be embryos with a period of about 5-6 days after fertilization.

The term “internal cell mass (ICM)” refers to a group of cells found in mammalian blastocysts that can generate embryos and potentially form embryos and all tissues other than embryonic embryos. In general, the internal cell mass will be a cell population in the blastocyst from which human ES cells are derived.

The term "cell" as used herein also refers to individual cells, cell lines or cultured cells derived from such cells or cell lines. The term "cell line" as used herein refers to an ES cell or a cell derived from an ES cell, such as maintained in in vitro culture. In general, the cells or cell lines of the invention or the cells or cell lines used in the methods of the invention are human cells or human cell lines.

The term "human ES cell" refers to a cell derived from a human embryo that exhibits a pluripotent phenotype as defined below.

The term "pluripotency" refers to a cell or cells that have the potential for development to differentiate into a range of differentiated cell types. Preferably, pluripotent cells have the potential to differentiate into all three derivatives of the endoderm, mesoderm and ectoderm of the embryonic germ layer.

The term "medium" refers to a suitable medium capable of maintaining undifferentiated ES cells. Similarly, "medium capable of maintaining undifferentiated embryonic liver cells" refers to a composition in which ES cells can proliferate, maintain a normal karyotype, and maintain pluripotency. Various such media are known in the art and are described below.

The term "follicular fluid" refers to a liquid which, during follicular growth, facilitates the transport of nutrients from the plasma to nourish the egg. In general, the follicular liquid used in the method of the present invention is a human follicular liquid (hereinafter referred to as “hFF”).

In one aspect, the present invention provides undifferentiated human ES cells capable of proliferation in vitro and methods of producing such cells. Methods for producing undifferentiated ES cells include fusion of cryopreserved human embryos and culturing some or all of the embryos on a medium capable of maintaining undifferentiated embryonic liver cells. Preferred frozen human embryos for use in this fusion step are cryopreserved embryos in blastocysts. Most preferred are blastocyst embryos that are cryopreserved about 5-6 days after in vitro fertilization. In the present invention, it is also possible to use human blastocyst embryos that have been frozen for more than several years. For example, human conjugates (ie, fertilized eggs or embryos) used in embodiments of the present invention are blastocyst embryos that are generally discarded after storage for at least 4 years after in vitro fertilization (human IVF-ET) is performed. . In general, extra conjugates in infertility clinics are cryopreserved for subsequent implantation. However, the current policy of many infertility clinics is to discard the cryopreserved and stored conjugates after a period of time if there is no need for additional implantation from the parent or no further contact from the parent. An important feature of the present invention is the use of cryopreserved conjugates which are usually discarded after a period of time. The timing of discarding a conjugate that is no longer needed depends on the bylaws applied by the policy or infertility clinic.

In one embodiment of the present invention, a first step of treating a cryopreserved human blastocyst embryo in a solution comprising a follicular solution with a first solution comprising a follicular solution, preferably hFF and an antifreeze agent; A method of fusion of cryopreserved human embryos is provided by a subsequent second step of treating human blastocyst embryos cryopreserved with a second solution containing a lower concentration of anti-freeze agent than the first solution. Preferably, the second solution also includes a follicular liquid.

In one embodiment of the invention, about 5-6 days old blastocyst embryos are exposed to a follicular fluid, preferably a solution containing hFF, followed by an East Sea like glycerol or sucrose to prevent embryo damage and maintain adequate osmotic pressure. Exposure to solutions containing inhibitors. In a preferred embodiment, the antifreeze solution also comprises a follicular liquid, preferably hff. The embryo is then frozen and stored cryogenic in liquid nitrogen. After a period of time, preferably after several years of storage of frozen fertilized eggs, the method of the invention can be used to establish ES cells from embryos. For example, the methods of the present invention can be used to establish ES cells from cryopreserved embryos for at least 4 years.

In another embodiment of the present invention, a method of establishing undifferentiated human ES cells comprises obtaining a population of cryopreserved human embryos during blastocyst development; Fusing one or more embryos; And culturing some or all of each of the fused embryos on a medium capable of maintaining undifferentiated ES cells. In this embodiment, the population refers to a collection of two or more embryos, in particular two or more embryos in the blastocyst stage. As disclosed herein, blastocysts have a higher likelihood of success in establishing undifferentiated human ES cells than embryos in the early stages of development. Thus, the present invention is useful in a method in which most or all of the stored embryos, preferably all of the stored embryos, are in the blastocyst.

At the time of fusion of human embryos, anti-freeze agents should be removed to protect the embryos. The present invention is characterized in that it is possible to reduce the concentration of the anti-inflambicide at various stages and to process the follicle solution to efficiently remove the anti-thaw agent from cryopreserved embryos. In a preferred embodiment, the embryo is maintained in follicular fluid throughout the process of removing the anti-inflammatory agent. Only surviving embryos are cultured in cell culture. This step is a modification of existing techniques, for example, Menezo et al. (Menezo et al., 1995), which achieves high stability by efficiently removing the anti-dong agent while maintaining an appropriate osmotic pressure. do.

When dissolving frozen fertilized eggs, various methods can be used, such as those reported by Menezo. Such methods are described in Menezo YJR et al., 1992, Human Reproduction 7: 101-106; Kaufnmann R et al., 1995, Fert. Steril., 64: 1125-1129; Menezo YJR et al., 1996, Abstract ASRM 52nd Annual Meeting . The melting method of the present invention is very simple, easy and efficient compared to the Menezo method in terms of the number and time of treatment steps. For example, in one embodiment, the melting method requires 5 steps and 17 minutes, whereas The setup requires seven steps and a minimum of 38 minutes. Reduction of treatment steps and time for fusion prevents unnecessary embryo damage from in vitro environmental exposure.

According to the invention, fusion takes place using two or more solutions containing different concentrations of antifreeze agents. Thus, the melting method may comprise two, three, four, five or more steps. Preferably, the melting method will require up to five steps. As will be appreciated by those skilled in the art, the number of steps in the melting method can be optimized according to the target survival rate of the fused embryo. In addition, the duration of each stage can vary depending on the desired survival rate and can vary from a short time (ie less than about 2 minutes) to a long time (about 10 minutes or more). Preferably, the one or more steps will last about 2 to 10 minutes, more preferably about 4 to 6 minutes, generally about 5 minutes.

The anti-freeze agent used in the melting method of the present invention can be any compound known to inhibit the crystal formation of water at freezing temperatures, especially compounds that do not significantly reduce the survival of the embryo. "Severely reducing" means reducing the survival rate by at least 30%, preferably at least 20%. Such antifreeze agents are known in the art and include various compounds such as sugars such as sucrose, glucose and the like; Polyols such as polyethylene glycol; And various organics such as ethylene glycol, methyl pentanediol, glycerol and the like. The concentration of antifreeze agent used depends in part on the method used to freeze the embryo. For example, if a slow freezing method has been performed, a relatively small amount of antifreeze agent is used. Therefore, when melting the embryo, a small amount of anti-freeze agent will generally be used. If the freezing method used a relatively large amount of antifreeze agent, the concentration of the antifreeze agent used in the melting method would be greater. In general, the concentration of antifreeze agent will be in the range of about 10-50% depending on the freezing method used in cryopreservation of the embryo. In the present invention, preferably, the concentration of the antifreeze agent used in the melting step will be 30% or less, more preferably 20% or less, most preferably 10% or less. For example, glycerol may be used at a concentration of 9% and sucrose at 0.4M (about 14%).

Methods of slowing and freezing common embryos are known in the art. These methods are described in Rall, W.F. and Fahy, G. M., 1985, Nature, 313: 573-575; Gordts S. et al., 1990, Fertility Sterility 53 (3): 469-472; Fahning, ML and Garcia, MA, 1992, Cryobiology, 29: 1-18; Abbeel van den E. et al., 1997, Human Reproduction, 12 (7): 1554-1560. Thus, those skilled in the art will be able to modify methods known in the art in accordance with the teachings of the present invention to achieve improved methods of freezing and thawing embryos, and improved methods of establishing undifferentiated human ES cells. Could be.

Continuous antifreeze solutions generally have a decreasing amount of antifreeze agent. For example, the glycerol containing solution may be reduced in concentration by about 2% per continuous solution. The sucrose-containing solution may be reduced by about 0.2 M (about 7%) per continuous solution. Beyond the scope of the examples mentioned above, any further changes may be present and may be determined by one of ordinary skill in the art.

In addition, multiple anti-corrosive agents may be used in one solution. For example, one solution may include both glycerol and sucrose. Generally, the combined concentration of anti-inflammatory agents does not exceed about 30%. In a preferred embodiment, the solution comprises about 5% glycerol and about 0.4 M sucrose. The concentration of only one antifreeze agent, two or more antifreeze agents or all antifreeze agents in a continuous solution may be reduced.

In a preferred embodiment, the invention features the use of human follicular fluid in the thawing of fused fertilized eggs or embryos. The follicular fluid contains all the essential nutrients and / or environmental conditions for the human embryo or fertilized egg to grow, and also contains the growth and inhibitory factors necessary for the embryo to survive. Thus, the use of hFF in the fusion process offers the advantage of an improvement in embryonic persistence in subsequent culture steps of the embryo, for example in the culture of the embryo to establish ES cells.

In a preferred embodiment, the method of thawing cryopreserved human embryos comprises two or more solutions comprising hFF and antifreeze agent. In a preferred embodiment of the invention, the two or more solutions comprise about 15-25% by volume of hFF.

In another preferred embodiment of the invention, the fusion of human embryos comprises three, each comprising about 4-6 volume percent glycerol, about 2-4 volume percent glycerol and about 0.1-2 volume percent glycerol and further each hFF Continuous use of the solution. Preferably, the one or more successive steps are performed for about 4-6 minutes. The treated embryos are then further washed with one or more solutions, preferably comprising 15-25% by volume of hFF.

In a method of forming ES cells from an embryo, the fused human embryo is enzymatically digested to remove the zona pellucida or part thereof. Any protein enzyme can be used to degrade the zona pellucida or a portion thereof from blastocyst. Various methods and enzymes known in the art to be useful for this degradation step can be used, including pronase and acid Tyrodes solutions. Preferably pronase is used. To resolve the zona pellucida, pronase is used for a time sufficient to remove the zona pellucida, which can be readily determined by one skilled in the art. Enzymatically treated embryos are further cultured to dissolve the zona pellucida and eliminate pronase toxicity. Removal of the zona pellucida exposes trophic ectoderm and internal cell mass (ICM).

According to the method of the present invention, the trophectoderm is isolated from the ICM, preferably in the process of treating the embryo by immunotomy. In certain embodiments of the invention, novel immunotomy is provided. In particular the use of anti human lymphocyte antibodies (hereinafter referred to as "AHLS") is another feature of the invention for removing trophectoderm. Thus, in order to effectively establish human ES cells from freeze-thawed blastocyst embryos, AHLS can be used in the immunoresection procedure.

Prior to the present invention, anti-human serum antibodies were generally used to remove trophectoderm. However, the inventors of the present invention have recognized that anti-human serum antibodies do not completely remove trophectoderm from ICM. The establishment of undifferentiated ES cells from ICM is highly dependent on the complete removal of trophectoderm from ICM. Thus, the use of AHLS according to the method of the present invention increases the efficiency of establishing undifferentiated ES cells from ICM.

Without wishing to be bound by theory, it is believed that AHLS is more effective than anti human serum antibodies based on the characteristics of lymphocytes. Lymphocytes are cells with antigen specificity that are predetermined to react only with specific antigens. Lymphocytes have adherent independent cell characteristics similar to embryonic cells. Thus, both cell types have similar cell surface antigens. AHLS with similar properties can be used to induce high antigen-antibody responses against embryos. Thus, the present invention presents a separate method of degrading trophectoderm using AHLS, which has been found to be more effective in degrading trophectoderm in immunoresection than other available antibodies. Therefore, in the present invention, in order to selectively obtain internal cell mass (ICM), AHLS was specifically designed for immunotomy.

As will be appreciated by those skilled in the art, AHLS can be obtained from a variety of animals, including mice, rats and other animals known to produce antibodies, for which methods of obtaining the desired antibodies are known. For example, AHLS can be obtained from human blood and injected into rabbits with human lymphocytes mixed with an adjuvant or Freund's adjuvant. Except that human lymphocytes are used as antigens, the desired antibodies can be obtained from rabbits using common methods available in the industry concerned.

Prior to immunotomy, complement inactivation by heating of AHLS is preferred. Exposure of complement inactivated AHLS provides rapid rupture of the embryo. Recommended temperatures in the serum inactivation protocol range from about 45 ° C. to about 62 ° C., and time ranges from about 15 to about 60 minutes. The most commonly used method is to treat serum at about 56 ° C. for about 30 minutes. Appropriate treatment concentrations and time of antibody and complement in immunoresection are very important for establishing ES cells. In conventional immunotomy, about 10% antibody and about 20% complement are generally used, but in the present invention, preferably, about 2-5% antibody is treated for about 30 minutes to 1 hour. And complement at a concentration of about 5-12% for about 40-60 seconds.

Thus, establishing ES cells includes removal of the zona pellucida and immunotomy in the fused embryo. The thawed embryos are treated with pronase to remove the zona pellucida and then incubated for a period of time to eliminate pronase toxicity. The culture is then exposed to AHLS to induce an antigen-antibody response and further treated with complement to completely degrade the trophoderm. Complement was obtained from whole serum and its type is important. In general, rabbit serum is not preferred because it may be toxic, and guinea pig serum or rat serum is more preferred. Depending on the survival rate of ICM after complement treatment, one with skill in the art may use other complements.

Thus, one embodiment of the present method for establishing human ES cells is capable of maintaining the undifferentiated ES cells and processing to remove embryonic or ectodermal embryos using AHLS to obtain embryonic ICM. Culturing at least a portion of the ICM on the medium.

The present invention also includes a method for separating ICM according to the above-mentioned AHLS immunoablation method. Preferably, the method of separating ICM further comprises cryopreserved and fused human blastocyst embryos.

The methods of the invention also include isolated ICMs of human blastocysts formed using the AHLS immunoresection methods of the invention mentioned above.

Cultivation of ICM can be performed using a variety of methods known in the art, including culturing ICM on fibroblast feeder cell layers. In order to separate ES cells from culture, the culture conditions should not induce differentiation and keep the cells in a proliferative state. For this purpose, various differentiation inhibitory factors must be added additionally. For example, mouse embryonic fibroblast (MEF) cells can be used as a trophic layer for ES cell culture to help maintain ES cells as pluripotent liver cells. Inhibition of differentiation of ES cells by MEF feeder cells appears to be due to the production of leukemia inhibitory factor (LIF) (Williams et al., 1988). MEF cells can be isolated directly from mouse embryos (14-16 day old embryos) or supplied as pre-made products (STO cells). In addition, it is desirable to add LIF in addition to STO cells to culture ICM. STO cells (# CRL-1503) can be purchased from American Type Culture Coollection (U.S.A.). STO cells are passaged every two days. In order to inactivate somatic cell division of feeder cells, it is preferable to drop the recovered cells after treatment with mitomycin-C.

The isolated ICM can be plated and grown in culture conditions suitable for human liver cells. Proliferation in vitro can include long term cell culture. The cells remain substantially in the undifferentiated state. Preferably, the cells are maintained under conditions that do not induce cell death or extravasation. Also preferably, they can maintain an undifferentiated state when cultured on a fibroblast trophoblast layer, preferably under conditions that do not differentiate. Any vessel known in the art to maintain ES cells may be used. For example, preferred media for maintaining undifferentiated ES cells include 20% fetal bovine serum (FBS), 1 mM glutamine, 0.1 mM β-mercaptoethanol, 1% ribonucleosides, 1% non-essential amino acids and 0.1% It consists of 80% Dulbecco's modified Eagle's medium (DMEM, no sodium citrate, high concentration of glucose preparation) supplemented with human LIF (2000 units / ml). After 7-10 days after initial plating, ICM induced exogenous growth was mechanically separated and plated again in fresh feeder cell layers. The resulting expanded colonies were mechanically separated about every 7 days and cut into chunks of 50-100 cell numbers.

In a preferred embodiment, the cells have the ability to differentiate in vitro when exposed to differentiation conditions. Most preferably, the cells have the ability to differentiate into a wide range of somatic systems in vitro.

Undifferentiated ES cells can be confirmed by characteristic morphology observation. Identification of these features is also possible by various methods known in the art. Methods such as microscopic examination of the form or measuring alkaline phosphatase activity can be used. Morphological features of ES cells are well known in the art and include international patents such as James Thompson et al. ( Science 282: 1145-1147), Rubinov et al . ( Nature 18: 399-404) and Gearheart et al. See WO 98/43679. Thomson and Gearheart et al. Described several forms of good primate ES cell lines, such as normal karyotypes and high levels of intracellular alkaline phosphatase (AP), and the presentation of specific cell surface glycolipids and glycoproteins that are not characteristic or specific for pluripotent liver cells. It is reported to have scientific characteristics. Other important features include growth as multicellular colonies, normal and stable karyotypes, the ability to be serially passaged, and the ability to differentiate into cells derived from all three types of embryonic germ cell layers. "Normal karyotype" means that the chromosome composition of a cell has normal characteristics in that species in terms of the number, size, and morphology of the medium chromosome.

For more accurate identification of ES cells, it is more desirable to measure alkaline phosphatase activity in conjunction with morphological studies. This method makes it possible to identify ES cells by observing the degree of staining. Only undifferentiated ES cells are stained and differentiated cells are not stained. FIG. 13 shows unstained differentiated cells and FIG. 14 shows stained ES cells.

Different expressions of Oct-4 can be used to identify undifferentiated ES cells and differentiated cells. Oct-4 is particularly strongly expressed in early embryonic and gonad and embryonic liver cells and is present as Oct-4a and Oct-4b by alternative splicing. Oct-4b is expressed more strongly than Oct-4a in ES cells (FIG. 27). In addition, Oct-4b is expressed in ES cell colonies (B, C and D; lanes 4 to 11) (FIG. 27), but in embryonic embryos (EB) (A; lanes 1 to 3) (FIG. 27) or differentiated colonies. It is not expressed in (FIG. 28). FIG. 27 shows electrophoresis results showing different expression levels of Oct-4 in embryonic bodies (EB) and ES cell colonies. Lane A in Fig. 27 shows embryonic bodies, lanes B to D represent ES cells. 28 shows electrophoresis results showing different expression levels of Oct-4 in ES cells (S1, S2) and differentiated ES colonies (D1, D2). As shown in FIG. 28, Oct-4b is not expressed in differentiated ES cells, but Oct-4b is expressed in undifferentiated ES cells. Thus, expression of Oct-4 will be a potent marker of the presence of undifferentiated ES cells.

Differentiation of ES cells into specific cells can be induced by a variety of methods. Differentiation of ES cells in vitro occurs when the cells become able to aggregate in suspension media without STO feeder cells and LIF. Aggregates of ES cells are further differentiated into embryoid bodies, including various cell types, including cardiomyocytes, neurons and other cells. In addition, ES cells grown at high concentrations for a long time without replacing the feeder cell layers form aggregates and vesicular structures in which various types of differentiated cells are found. In the absence of growth factors, cells can spontaneously differentiate into many different types of colonies, while the addition of growth factors can produce more mature cell types. Cultures treated with growth factors are more homogeneous, and more than half of the cultures may contain one or two cell types (muscle-like synitiums in actividin-A treated cells, RA treatment Neuronal like cells in cultured cultures, and fibroblast-like cells in BMP-4 treated cultures, etc.). In addition, pluripotent liver cells express a wide range of growth factor receptors and can enrich many human cell types in vitro by specific factors (Shuldiner et al., 2000).

Effective preservation of ES cells is very important as it permits continuous storage of cells for later use. Commonly used methods for cryopreservation of cell lines can be used. However, an efficient method for cryopreservation of embryos is most appropriate for this purpose. According to the invention, colonies can be frozen at high speed using ES culture medium to which 10% DMSO is added. In one embodiment, it may be important to slowly cool the cells to prevent the formation of ice crystals within the cells. For example, cells may be frozen at −20 ° C. for 1 hour, then transferred overnight to the −70 ° C. region, and then transferred to liquid nitrogen the following day.

Recently, much attention has been given to the potential applications of liver cells in the biological and pharmaceutical fields. Pluripotency and immortality are specific to ES cells, and this property has made it possible for the first time for researchers to approach many problems in human biology and medicine. ES cells can potentially address the problem of lack of donor tissue for use in the transplantation process, particularly the absence of alternative culture systems that can support the growth of the committed stem cells required. ES cells have a very wide range of applications in areas such as human medicine, developmental research, functional genetics, identification of new growth factors and drug discovery and toxicology.

The invention will be described in more detail through the following examples, which are not intended to limit the scope of the invention.

As the cells, compositions, reagents, methods or uses are naturally variable, the present invention is not limited to the described cells, compositions, reagents, methods or uses. The terminology used herein is for the purpose of describing particular embodiments only, and the terminology used herein is not intended to limit the scope of the invention. Throughout the specification, the preferred embodiments and examples presented should be considered as examples only, rather than as limiting the invention. All of these will be apparent to those skilled in the art after retaining the benefit of this disclosure.

Example

Example 1 Cryopreservation of Embryonic Embryos

Extra 5-6 day blastocyst embryos produced from a human in vitro baby program at Maria's Hospital were exposed to a solution containing 20% human follicular fluid (hFF) for 10 minutes, followed by a solution containing 20% hFF and 5% by volume glycerol. Treatment was again performed for 10 minutes. The embryos were then treated for 10 minutes with a solution containing 20% hFF, 9 vol% glycerol and 0.2 M sucrose. Programmed freezing curves were selected from 22 ° C. to −7 ° C. at a rate of −1 ° C./min using a programmed biological freezer (Planar Kryo: T.S. Scientific, Perkasie, PA). After a 30 second delay, seeding was done by hand. After seeding, the embryos were slowly cooled from -7 ° C to -40 ° C at a rate of -0.3 ° C / min. Thereafter, it was immersed in liquid nitrogen.

Example 2: Fusion of Frozen Embryonic Embryos

After thawing frozen embryos treated as described above and cryopreserved for at least 4 years, the fused embryos were treated with a solution containing 20% hFF, 5 vol% glycerol and 0.4 M sucrose, followed by 20% hFF, 3 The solution was treated for 5 minutes with a solution containing volume% glycerol and 0.2 M sucrose. It was then again treated for 5 minutes with a solution containing 20% hFF, 1 volume% glycerol and 0.2 M sucrose, followed by exposure for 2 minutes to a solution containing 20% hFF and 0.2 M sucrose. Thereafter, the embryos were treated with a solution containing 20% hFF for 5 minutes (FIG. 1), and the surviving embryos were incubated in the culture medium for 24 hours (FIG. 2).

Example 3: Preparation of Rabbit Anti-Human Lymphocyte Antibodies (AHLS)

Human lymphocytes (> 4 × 10 ⁸ cells) recovered from a study grade Ficoll plaque (Amersham Pharmacia Biotech AB) treatment were treated with RIBI adjuvant (R-700, RIBI Immunome Research (R-700). Purchased from Immunochem Research, Inc.) or Freund's adjuvant and rabbits were injected a total of four times at two week intervals. AHLS was recovered from the rabbit's heart 10 days after the last injection.

Example 4: ICM Isolation Using Immunotomy

Prior to immunotomy, AHLS was inactivated by heat complement at 56 ° C. for 30 minutes. Complement was absorbed with agarose, stored at -70 ° C and melted immediately before use. Complement acting on the cell part induced the antigen-antibody response to block the action. The 5% antibody was treated for 30 minutes and the 10% complement was treated for 40-60 seconds. After dissolving the zona pellucida with TL-Hepes solution containing 0.25% pronase (Figure 3), it was exposed to 5% rabbit anti-human lymphocyte antibody (AHLS) for 15 minutes and 10% guinea pig complement ICM was separated by exposure to 1-2 minutes (FIG. 4).

Example 5: Preparation of STO Cells

STO cells (mouse embryonic fibroblasts, ATCC) were prepared for co-culture with internal cell masses. Frozen STO cells were exposed to air for 20 seconds and placed in warm water (36.5 ° C.) until the cells were completely fused. 9 ml warm HBSS or STO culture medium was added and mixed. After centrifugation at 1000 rpm for 10 minutes, the supernatant was removed and the precipitate was resuspended in 3 ml warm DMEM (Dulbecco's modified Eagle's medium) with 10% FBS and incubated in a 25-T culture flask. The resulting cultures were passaged once every two days.

Example 6: Preparation of feeder drop cells from isolated STO cells

After confirming the confluence of STO cells in the culture flask, the cells were washed with PBS solution, treated with DMEM with 10 μg / ml of mitomycin C, and treated with DMEM for about 2 hours and 30 minutes, followed by 3 times with PBS solution. Washed and then treated with 1 × Trypsin-EDTA. The resulting cells were washed with warm 5 ml HBSS solution and centrifuged for 5 min at 1000 rpm. After discarding the supernatant, the precipitate was resuspended in 5 ml of HBSS solution. The solution was again centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded and the precipitate was resuspended in 1 ml of DMEM-LIF solution. Then, some of the cells were counted using trypan blue (1: 1) and hematocytometer counting chambers, and the remaining cells were resuspended and then inoculated in culture plates (250,000 cells / ml). It was.

Example 7: Culture and Passage Culture of ICM Cells

The isolated ICM cells were placed on STO cell trophoblasts using a micro pipette (FIG. 7) and continuously incubated with daily replacement with fresh DMEM solution containing 2000 units / ml LIF. The formed ICM mass was re-coated on a fresh STO nutrient layer and continuously incubated. The ICM masses were mechanically separated and divided into several lower colonies and incubated on a new STO nutrient layer about once every seven days.

Example 8: Characterization of ES Cells

According to methods published by James A. Thompson et al. (1998, Science 282: 1145-1147) and Benjamin Rubinov et al. (2000, Nature 18: 399-404), ES cells were identified by morphological observation under a microscope. . In addition, the ES cells were fixed for 15 minutes in 4% formaldehyde, washed three times with sterile distilled water, and then, using Fast Red TR / Naphtol AS-MX solution purchased from Sigma (Sigma, St. Louis, MO). After staining for 30 minutes, the degree of staining was observed.

Alternatively, RT-PCR was performed on colonies consisting mostly of liver cells to measure the expression of Oct-4. mRNA was isolated via whole cell extraction and cDNA synthesis was performed by reverse transcription. Primers used for PCR amplification were 5'-CCACATCGGCCTGTGTATAT-3 '(antisense primers for Oct-4a and Oct-4b) and 5'-CTCCTGGAGGGCCAGGAATC-3' (sense primer for Oct-4a), 5'-ATGCATGAGTCAGTGAACAG -3 '(sense primer for Oct-4b). PCR amplification was performed 35 times for 1 minute at 94 ° C, 1 minute at 55 ° C, and 1 minute at 72 ° C. PCR products were visualized by ethidium bromide staining on a 1% agarose gel and then examined with an image analyzer (Biorad). In this way, the expression levels of Oct-4a and Oct-4b were measured, and the results are summarized in Table 1 below.

Number of fused blastocyst embryos 6 Number of blastocysts surviving after thawing 5 Number of blastocyst embryos subject to immunotomy 4 The number of ICMs plated on the badge 4 The number of ICMs attached to the medium 2 Number of colonies formed from ICM 2 Number of colonies formed after 5-6 passages 2

As shown in Table 1 above, four blastocyst embryos were obtained that could be used for immunotomy, and two colonies were formed from four ICMs. These two colonies were divided into several lower colonies, and most of the ICM continued to proliferate undifferentiated during five to six passages (45-50 days after immunotomy). In addition, the karyotypes of cell cultures derived from freeze-thawed human embryos were examined. For chromosome analysis, 10-15 generations of human ES colonies were incubated in dishes for 3-5 days without the STO trophic layer. 5% colsemid was treated and collected cells were stained by standard G-band techniques. Mid-term spreads and karyotypes were examined by a cytovision program (Applied Imaging Co. FIGS. 30 and 31). Alkaline phosphatase activity was also measured. Alkaline phosphatase activity was not detected in differentiated colonies (FIG. 13) There was strong phosphatase activity in undifferentiated colonies (Figure 14) In addition, the expression of Oct-4 was expressed in Oct-4b in ES cells but not in differentiated cell types, embryoid and differentiated colonies. It was confirmed (FIGS. 27 and 28).

Example 9: Differentiation of Human ES Cells into Specific Cells

To the cells identified above as embryonic liver cells, growth factors and basic culture medium (L-glutamine, non-essential amino acids and 20% FBS) were added to induce differentiation. To differentiate into cardiomyocytes, 1 μM of retinoic acid (RA) was added to the colonies during the differentiation process. The results are shown in FIGS. 15 to 17. To induce differentiation into neurons, 1 μM retinoic acid (RA), 10 ng / ml basic fibroblast growth factor (b-FGF), 100 ng / ml nerve growth factor NGF) was added. The culture was changed daily. In addition, differentiation into muscle cells was confirmed under differentiation conditions to the cardiomyocytes.

Example 10 Confirmation of Differentiation into Specific Cells

Differentiation from ES cells to specific cells was primarily identified by their characteristic morphological characteristics. In the case of differentiation into cardiomyocytes, as shown in FIG. 17, morphological features showing a routine heart-beat were observed. In addition, differentiated cells were identified by immunocytochemical method. To identify neurons, specific monoclonal antibodies were used to detect 200 kDa neurofibrillary protein (NF200) (Sigma), microtubule binding protein 2 (MAP2) (Sigma), and β-tubulin (Sigma). . The results are shown in FIGS. 19 to 21. In addition, in order to identify glial cells, neuroglial fibrosis protein (GFAP) and galactoselebroseside (GalC) were detected using polyclonal antibodies. The results are shown in FIGS. 22 to 24. Muscle cells were identified with monoclonal antibodies having specificity for muscle actin (Sigma) (FIGS. 25 and 26). In order to confirm the degree of response to each antibody, secondary antibodies with FITC were prepared. The whole procedure for staining was carried out at 4 ° C. This method is based on Rubinov et al.

Example 11: Cryopreservation and Thawing of Human ES Cells

Cryopreservation was attempted to confirm the long term storage of human ES cells. 10% DMSO was added to the ES cell medium to prepare an antifreeze agent for cryopreservation of ES cells, and used after filtration. In order to reduce the toxicity of the anti-inflammatory agent, colonies of ES cells were introduced directly into a freezing tube containing the anti-inflammatory agent and placed at -20 ° C for 2 hours. The frozen cells were then stored at −70 ° C. for 10 months.

The freeze tube was thawed completely in a 36 ° C. water bath and the contents of the tube were placed in ES culture medium to remove the anti-inflammatories. The isolated ES cell mass was quickly placed in the culture vessel and the viability was examined. It has been shown that ES cells produced by the present invention can survive even after freeze thawing. 29 shows this result.

Although the present invention has been described above, it will be apparent that many other variations and modifications are possible while maintaining the advantages of the present disclosure to those skilled in the art. All such modifications and variations are considered to be within the scope and spirit of the invention.

Embryonic liver cells can be effectively obtained from long-term frozen 5-6 day blastocyst embryos using the method of establishing embryonic liver cells from freeze-thawed human embryos of the present invention. The embryonic stem cells thus obtained can be widely used in early human embryo research, as well as in the development of new growth factors and medicines, in the treatment of diseases and transplantation therapy.

Claims (26)

(a) fusion of cryopreserved human blastocyst embryos, and (b) culturing some or all of the embryos on a medium capable of maintaining undifferentiated embryonic liver cells to establish undifferentiated human embryonic liver cells Including, the method of establishing undifferentiated human embryonic liver cells. The method of claim 1, wherein the blastocyst embryo comprises a sphere of cells having an outer cell layer, a liquid packed cavity and an inner cell mass. The method of claim 1, wherein the blastocyst embryo is cryopreserved 5-6 days after fertilization of the embryo. The method of claim 1, wherein the human blastocyst embryo is cryopreserved for at least 4 years. The method of claim 1, wherein said melting step (a) a first step of treating said cryopreserved human blastocyst embryo with a first solution comprising an anti-inflammatory agent and a human follicle solution, and (b) treating the cryopreserved human blastocyst embryo with a second solution comprising a reduced concentration of anti-freeze agent and human follicular fluid compared to the first solution. 6. The method of claim 5, wherein said anti-inflammatory agent is selected from the group consisting of sucrose, glycerol and a combination of sucrose and glycerol. The method of claim 1, wherein said melting step (a) a first step of treating said cryopreserved human blastocyst embryo with a first solution comprising an anti-inflammatory agent and a human follicle solution, (b) a subsequent second step of treating said cryopreserved human blastocyst embryo with a second solution comprising a reduced concentration of antifreeze agent and human follicle fluid compared to said first solution, (c) a subsequent third step of treating said cryopreserved human blastocyst embryo with a third solution comprising a reduced concentration of antifreeze agent and human follicular fluid as compared to said second solution, (d) treating the cryopreserved human blastocyst embryo with a fourth solution comprising a reduced concentration of anti-freeze agent and human follicle fluid compared to the third solution The method consists of. 6. The method of claim 5, further comprising a subsequent third step of treating said cryopreserved human blastocyst embryo with a third solution comprising an anti-inflammatory agent and a human follicle solution, wherein the third solution is about 0.1 to 2% by volume. Glycerol, and wherein the second solution comprises about 2 to 4 volume percent glycerol and the first solution comprises about 4 to 6 volume percent glycerol. The method of claim 5, wherein at least one of the continuous steps is performed for about 4 to 6 minutes. The method of claim 5, wherein the first solution and the second solution each comprise about 15-25% of human follicular fluid. The method of claim 1, further comprising the step of removing trophoblastic cells from said embryo using an anti human lymphocyte antibody. The method of claim 1, wherein the portion of the embryo comprises an internal cell mass. Undifferentiated human embryonic liver cells formed using the method of any one of claims 1-12. (a) obtaining a population of cryopreserved human embryos consisting solely of blastocysts, (b) melting one or more of the embryos, and (c) culturing some or all of each of the fused embryos in a medium capable of maintaining undifferentiated embryonic liver cells and establishing undifferentiated human embryonic liver cells Method for establishing undifferentiated human embryonic liver cells comprising a. (a) a first step of treating cryopreserved human blastocyst embryos in a solution comprising human follicle fluid with a first solution comprising an anti-inflammatory agent and a human follicle fluid, (b) at least one subsequent second step of treating the cryopreserved human blastocyst embryo with a second solution comprising a reduced concentration of anti-freeze agent and human follicle fluid compared to the first solution Method of melting the cryopreserved human embryo comprising a The method of claim 15, wherein said embryo is blastocyst embryo. The method of claim 15, wherein the anti-inflammatory agent is selected from the group consisting of sucrose, glycerol and a combination of sucrose and glycerol. The method of claim 15, wherein the subsequent step is (c) treating the cryopreserved human blastocyst embryo with a third solution comprising a reduced concentration of antifreeze agent and human follicle fluid compared to the second solution, and (d) treating the cryopreserved human blastocyst embryo with a fourth solution comprising a reduced concentration of anti-freeze agent and human follicle fluid compared to the third solution The method consists of. The method of claim 15, further comprising the subsequent third step of treating the cryopreserved human blastocyst embryo with a third solution, the third solution comprising about 0.1-2% by volume of glycerol, and wherein the second The solution comprises about 2-4 volume percent glycerol and the first solution also comprises an antifreeze agent and a human follicular solution comprising about 4-6 volume percent glycerol. The method of claim 15, wherein at least one step of the continuous step is performed for about 4-6 minutes. The method of claim 15, wherein said first solution and said second solution each comprise about 15-25% human follicular fluid. A method of isolating internal cell mass of a human blastocyst embryo, comprising processing a human blastocyst embryo using an anti-human lymphocyte antibody. The method of claim 22, wherein the embryo is cryopreserved and fused. Undifferentiated human embryonic liver comprising culturing all or a portion of the internal cell mass obtained by the method of claim 22 or 23 on a medium capable of maintaining undifferentiated embryonic liver cells to establish undifferentiated human embryonic liver cells How to establish a cell. An isolated internal cell mass of human blastocyst formed using the method of claim 22 or 23. Undifferentiated human embryonic liver cell culture formed using the method of claim 24.