Method for evaluating multipotency of mammalian cells

Abstract

To provide a method capable of evaluating the multipotency of mammalian cells without using experimental mammals, such as immunodeficient mice, that can reproduce results from teratoma formation tests employing the experimental mammals, that has better cost effectiveness and time effectiveness than cases employing the experimental mammals, and that consists of relatively simple procedures.SOLUTION: A method of evaluating the multipotency of mammalian cells comprises the following steps: (a) transferring the mammalian cells onto a chorioallantoic membrane of an embryonated egg, and then culturing the embryonated egg; and (b) evaluating the multipotency of the mammalian cells using, as an indicator, teratoma formation in the embryonated egg cultured in the step (a).SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for evaluating the pluripotency of test mammalian cells by carrying out a teratoma formation test using embryonated chicken eggs. [Background technology]

[0002] In recent years, the rapid development of pluripotent stem cell research has led to a growing interest in regenerative medicine, and knowledge and understanding of the field is becoming more widespread not only among researchers but also among the general public. Regenerative medicine using pluripotent stem cells aims to restore the functions of cells and tissues damaged by various diseases by utilizing the self-renewal and multi-differentiation abilities of pluripotent stem cells, as well as factors secreted by pluripotent stem cells.

[0003] Methods for evaluating the quality of pluripotent stem cells include, for example, evaluation based on cell morphology (Patent Document 1), evaluation based on gene expression analysis (Patent Document 2), evaluation based on surface antigens, and the like. In addition, a teratoma formation test using immunodeficient mice has become an international standard for evaluating the pluripotency of embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells). When mammalian cells injected subcutaneously or into the testes of immunodeficient mice have pluripotency, teratomas differentiated into triploid tissues consisting of ectoderm, mesoderm, and endoderm are observed in the subcutaneous tissue or testes after a certain period of time has passed since the injection. On the other hand, teratoma formation tests using immunodeficient mice have problems in terms of cost performance, simplicity, and animal welfare, and a method for evaluating pluripotency without using experimental mammals such as immunodeficient mice has been desired.

[0004] The chorioallantoic membrane culture method of developing chicken eggs is known as a classic method for culturing tumor cells. It is also known that developing chicken eggs are used to culture and grow viruses to produce virus vaccines such as influenza virus and porcine deltacoronavirus (Patent Document 3, Patent Document 4). Recently, it has been reported that mouse ES cells were injected into the peripheral vein of the blastoderm of a 2-day embryo (developing chicken egg on the 2nd day after the start of incubation) using a capillary to perform a teratoma formation test (Non-Patent Document 1). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2006 / 057444 Brochure [Patent Document 2] JP 2004-194655 A [Patent Document 3] Special Publication No. 2010-539918 [Patent Document 4] JP 2021-119750 A [Non-patent literature]

[0006] [Non-Patent Document 1] Dev Growth Differ. 2016 Feb;58(2):194-204. Summary of the Invention [Problem to be solved by the invention]

[0007] The objective of the present invention is to provide a method for evaluating the pluripotency of mammalian cells that can reproduce the results of teratoma formation tests using experimental mammals such as immunodeficient mice without using such experimental mammals, is more cost-effective and time-effective than the use of such experimental mammals, and can evaluate the pluripotency of mammalian cells through relatively simple procedures. [Means for solving the problem]

[0008] As mentioned above, Non-Patent Document 1 describes that mouse ES cells were injected into the peripheral vein of the blastoderm in an embryonic chicken egg on the second day after the start of incubation using a capillary, and a teratoma formation test was performed, but since the peripheral vein is very small, injecting mouse ES cells into the peripheral vein using a capillary requires skill. Also, it is difficult to distinguish between fertilized and unfertilized eggs in embryonic chicken eggs on the second day after the start of incubation.

[0009] The present inventors have been conducting intensive research to solve the above problems, and in the process, have focused on the classical method of culturing on the chorioallantoic membrane of embryonated chicken eggs. In a preliminary experiment, melanoma cells (B16F10 cells) were injected into the allantoic cavity or chorioallantoic membrane of developing chicken eggs on the 10th day after the start of incubation and cultured for 2, 4, or 6 days, and the engraftment rate of B16F10 cells was analyzed. The engraftment rates when injected into the allantoic cavity were 0% (0 / 12), 33.3% (2 / 6), and 16.7% (1 / 6), respectively, whereas the engraftment rates when injected onto the chorioallantoic membrane were 53.3% (16 / 30), 74.2% (23 / 31), and 90.3% (28 / 31), respectively. This indicates that the engraftment rate of B16F10 cells was high when injected into the chorioallantoic membrane (rather than the allantoic cavity), especially when the developing chicken eggs were cultured for 6 days after injection.

[0010] Therefore, a diploid mouse ES cell line, which is a normal mouse ES cell line, was injected onto the chorioallantoic membrane of an embryonated chicken egg on the 10th day after the start of incubation, and the embryonated chicken egg was cultured for 6 days, after which the egg survival rate and the teratoma formation rate were measured. As a result, it was found that when the present teratoma formation method on the chorioallantoic membrane was carried out, a decrease in the egg survival rate and a decrease in the teratoma formation could be suppressed and the reproducibility of the teratoma formation was high, compared with the method described in Non-Patent Document 1 (i.e., a method in which a mammalian cell is injected into the peripheral vein of an embryonated chicken egg on the 2nd day after the start of incubation, and the embryonated chicken egg is cultured to confirm the teratoma formation). Furthermore, a teratoma formation test using immunodeficient mice was carried out using a tetraploid mouse ES cell line, which is known to have a low teratoma formation ability, and as a result, it was confirmed that the teratoma formation ability of the tetraploid mouse ES cell line was lower than that of the diploid mouse ES cell line, as in the result of the teratoma formation test using immunodeficient mice. The present invention has been completed based on these findings.

[0011] That is, the present invention is as follows. [1] A method for evaluating the pluripotency of a mammalian cell, comprising the following steps (a) and (b): (a) transferring mammalian cells onto the chorioallantoic membrane of an embryonated chicken egg and then culturing the embryonated chicken egg; (b) evaluating the pluripotency of the mammalian cells using teratoma formation in the embryonated chicken eggs cultured in step (a) as an indicator; [2] The method according to [1] above, wherein in step (a), the mammalian cells are transferred onto the chorioallantoic membrane of an embryonated chicken egg on or after the 10th day after the start of incubation. [3] The method according to [1] or [2] above, wherein in step (a), the embryonated chicken eggs are cultured for 6 days or more. [4] The method according to any one of [1] to [3] above, wherein in the step (a), the mammalian cells are injected onto the chorioallantoic membrane of an embryonated chicken egg using a syringe.

[0012] In another embodiment of the present invention, The above steps (a) and (b), and (p) administering the mammalian cells (pluripotent stem cells) evaluated to have high pluripotency in the step (b) to a subject in need of transplantation of the pluripotent stem cells (e.g., a patient with a traumatic disease, a patient with a tissue degenerative disease, or a patient with cancer). The following can be mentioned. Effect of the Invention

[0013] According to the present invention, a teratoma formation test using an experimental mammal can be reproduced by transferring mammalian cells onto the chorioallantoic membrane of an embryonated chicken egg by a relatively simple operation and culturing the embryonated chicken egg. In this way, the method of forming a teratoma on the chorioallantoic membrane of an embryonated chicken egg does not require the use of an experimental mammal, which has a higher unit price and maintenance cost than an embryonated chicken egg, and furthermore, since teratoma formation can be confirmed in a relatively short period of time compared to the case of a teratoma formation test using an experimental mammal, it is excellent in cost-effectiveness and time-effectiveness. Furthermore, the present method of forming a teratoma on the chorioallantoic membrane can suppress a decrease in egg survival rate and a decrease in teratoma formation compared to the method described in Non-Patent Document 1 (i.e., a method in which mammalian cells are injected into the peripheral vein of an embryonated chicken egg on the second day after the start of incubation and the embryonated chicken egg is cultured to confirm teratoma formation), and does not require skill when transferring mammalian cells to the embryonated chicken egg on the chorioallantoic membrane, which is larger than the peripheral vein. Therefore, the present invention contributes to the quality evaluation (multipotency evaluation) of pluripotent stem cells used in regenerative medicine, etc. [Brief description of the drawings]

[0014] [Figure 1] A schematic diagram of mammalian cells (ES cell line) injected onto the chorioallantoic membrane using a syringe. In the figure, "A" indicates the eggshell, "B" indicates the eggshell membrane, "C" indicates the chorioallantoic membrane, and "D" indicates the hole (slit). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The method of the present invention is not particularly limited as long as it is a method for evaluating the pluripotency of mammalian cells, which sequentially comprises: a step (a) of transferring test mammalian cells onto the chorioallantoic membrane of an embryonic chicken egg (i.e., a fertilized chicken egg) and then culturing the embryonic chicken egg (i.e., "an embryonic chicken egg with mammalian cells transferred onto the chorioallantoic membrane"); and a step (b) of evaluating the pluripotency of the mammalian cells using teratoma formation in the embryonic chicken egg cultured in step (a) as an indicator (herein, it may be referred to as "the present method for forming teratoma on the chorioallantoic membrane"), and does not usually include a step of culturing the mammalian cells in a site in the embryonic chicken egg other than the chorioallantoic membrane (e.g., in the allantoic cavity, in the amniotic cavity, in the yolk sac, in a blood vessel, in an embryo, in the blastoderm, or in a peripheral vein).

[0016] The above-mentioned mammalian cells may be any mammalian cell for which pluripotency needs to be evaluated, and examples thereof include mammalian cells of which it is unclear whether they have pluripotency (in other words, mammalian cells of which it is unclear whether they are pluripotent stem cells) and pluripotent stem cells of which it is unclear whether they have high pluripotency.

[0017] As used herein, "pluripotent stem cells" refer to cells derived from mammals that cannot become an individual by themselves but have the ability to differentiate into all tissues and cells that make up a living organism. Thus, pluripotent stem cells are different from multipotent stem cells (i.e., cells that have the ability to differentiate into multiple types of tissues and cells, but not all types) and unipotent stem cells (cells that have the ability to differentiate into a specific tissue or cell).

[0018] Examples of pluripotent stem cells include embryonic stem cells (ES cells), EG cells (embryonic germ cells), and induced pluripotent stem cells (iPS cells). ES cells can be produced by culturing inner cell masses on feeder cells or in a medium containing LIF. Methods for producing ES cells are described in, for example, WO96 / 22362, WO02 / 101057, US5,843,780, US6,200,806, US6,280,718, and the like. EG cells can be produced by culturing primordial germ cells in a medium containing mSCF, LIF, and bFGF (Cel11,70:841-847,1992). iPS cells can be produced by introducing reprogramming factors such as Oct3 / 4, Sox2, and Klf4 (and optionally c-Myc or n-Myc) into somatic cells (e.g., fibroblasts, skin cells, etc.) (Cel1, 126:p.663-676, 2006; Nature, 448:p.313-317, 2007; Nat Biotechno1, 26;p,101-106, 2008; Cel1 131:p.861-872, 2007; Science, 318:p.1917-1920, 2007; Ce11 Stem Cells 1:p.55-70, 2007; Nat Biotechnol, 25:p.1177-1181, 2007; Nature, 448:p.318-324, 2007; Cell Stem Cells 2:p.10-12, 2008; Nature 451:p.141-146, 2008; Science, 318:p.1917-1920, 2007).Stem cells established by culturing early embryos produced by nuclear transfer of somatic cell nuclei are also preferred as pluripotent stem cells (Nature, 385,810(1997); Science, 280, 1256(1998); Nature Biotechnology,17, 456(1999); Nature, 394,369(1998); Nature Genetics, 22, 127(1999); Proc. Nat1. Acad. Sci. USA, 96, 14984(1999)), Rideout III et al. (Nature Genetics, 24, 109(2000)).

[0019] In this specification, examples of "mammals" include rodents such as mice, rats, hamsters, guinea pigs, etc.; lagomorphs such as rabbits; ungulates such as pigs, cows, goats, horses, sheep, etc.; carnivores such as dogs and cats; and primates such as humans, monkeys, rhesus monkeys, cynomolgus monkeys, marmosets, orangutans, chimpanzees, etc., and among these, preferred examples include mice, pigs, and humans.

[0020] In the above step (a), the developing eggs for transferring the mammalian cells may be developing eggs in which the chorioallantoic membrane is formed, and considering that the chorioallantoic membrane is usually formed around the sixth day after the start of incubation, developing eggs on the sixth day or later after the start of incubation are preferred, developing eggs on the seventh day or later after the start of incubation are more preferred, developing eggs on the eighth day or later after the start of incubation are even more preferred, developing eggs on the ninth day or later after the start of incubation are even more preferred, and developing eggs on the tenth day or later after the start of incubation are preferably exemplified because the effect of the developing eggs is demonstrated in the present embodiment described later. In addition, considering that the development period of developing eggs is usually about 20 days after the start of incubation, developing eggs on the 20th day or earlier after the start of incubation are preferred, developing eggs on the 18th day or earlier after the start of incubation are more preferred, developing eggs on the 16th day or earlier after the start of incubation are even more preferred, developing eggs on the 14th day or earlier after the start of incubation are even more preferred, and developing eggs on the 12th day or earlier after the start of incubation are the most preferred.

[0021] In this specification, "incubation" means culturing an embryonated egg under conditions that allow normal embryo development, and the temperature conditions are usually within the range of 33°C to 38°C (preferably 35 to 37°C), and the humidity conditions are usually within the range of 40 to 60% (preferably 45 to 55%). Note that in this specification, the number of days after the start of incubation is counted as day 0, not including the first day, when the incubation is started.

[0022] In the above step (a), examples of the method for transferring mammalian cells onto the chorioallantoic membrane of an developed chicken egg include a method for transferring mammalian cells onto the chorioallantoic membrane of an developed chicken egg using a capillary (a capillary tube made of glass, synthetic resin, or the like), a polypropylene micropipette tip, a syringe, or the like (more specifically, a method for aspirating an isotonic solution containing mammalian cells into a capillary, a polypropylene micropipette tip, a syringe, or the like, making a hole [slit] in part of the shell of the developed chicken egg, and transferring the isotonic solution containing the mammalian cells onto the chorioallantoic membrane [e.g., by dropping, releasing, injecting, or the like]). From the viewpoint of simplicity, a preferred example is a method for injecting mammalian cells onto the chorioallantoic membrane of a developed chicken egg using a syringe. In this specification, the term "isotonic solution" refers to a solution in which the salt concentration, sugar concentration, etc. have been adjusted using sodium ions, potassium ions, calcium ions, etc. so that the osmotic pressure is approximately the same as that of body fluids or cellular fluids. Specific examples of isotonic solutions include physiological saline, physiological saline with a buffer effect (e.g., phosphate buffered saline (PBS), Tris buffered saline (TBS), HEPES buffered saline), Ringer's solution, lactate Ringer's solution, acetate Ringer's solution, bicarbonate Ringer's solution, 5% glucose aqueous solution, basal medium for animal cell culture (e.g., DMEM, EMEM, RPMI-1640, α-MEM, F-12, F-10, M-199), isotonic agents (e.g., glucose, D-sorbitol, D-mannitol, lactose, sodium chloride), etc.

[0023] In the above step (a), the number of mammalian cells transferred onto the chorioallantoic membrane may be at least one, but in consideration of efficiency, two or more mammalian cells (mammalian cell population) are preferred. The number of cells in the mammalian cell population is, for example, 10 to 1 × 10 9 in the range of (preferably 1×10 2 ~1×10 8 in the range of 1×10 3 ~1×10 7 within the range of 5×10 3 ~5×10 5 (within the range).

[0024] In the above step (a), the culture conditions of the developing chicken egg with the mammalian cells transferred onto the chorioallantoic membrane may be any conditions under which the embryo development of the developing chicken egg proceeds normally, and the temperature is usually within the range of 33°C to 38°C (preferably 35 to 37°C), and the humidity is usually within the range of 40 to 60% (preferably 45 to 55%). The temperature and humidity can be kept constant using an incubator or the like. The culture time may be any time sufficient for teratoma formation to be observed, and examples of the culture time include 24 hours (1 day) or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 7 days or more, 8 days or more, 9 days or more, 10 days or more, etc., and 6 days or more can be preferably exemplified because the effect of the culture time has been demonstrated in the present embodiment described later. In addition, the upper limit of the culture time is, considering that the development period of an embryonated egg is, as mentioned above, usually about 20 days after the start of incubation, usually before the 20th day after the start of incubation (preferably before the 19th day after the start of incubation, more preferably before the 18th day after the start of incubation, and even more preferably before the 17th day after the start of incubation).

[0025] In the above step (b), "evaluating the pluripotency of the mammalian cells using teratoma formation as an indicator" means evaluating whether the mammalian cells transferred onto the chorioallantoic membrane of a developing chicken egg have pluripotency (in other words, whether the mammalian cells transferred onto the chorioallantoic membrane of a developing chicken egg are pluripotent stem cells) using the presence or absence of teratoma formation as an indicator, and / or evaluating whether the mammalian cells transferred onto the chorioallantoic membrane of a developing chicken egg have high pluripotency using the teratoma formation rate (also referred to as "teratoma formation efficiency") as an indicator. Therefore, the above "method for evaluating the pluripotency of mammalian cells" means a method for evaluating whether mammalian cells have pluripotency and / or a method for evaluating whether mammalian cells have high pluripotency.

[0026] If teratoma formation is observed in the above step (b), it can be evaluated that the mammalian cells transferred onto the chorioallantoic membrane of a developing chicken egg are pluripotent stem cells (or the mammalian cell population transferred onto the chorioallantoic membrane of a developing chicken egg contains pluripotent stem cells). Furthermore, if the teratoma formation rate of the mammalian cells (pluripotent stem cells) transferred onto the chorioallantoic membrane of a developing chicken egg is higher than the teratoma formation rate of the comparative mammalian cells (pluripotent stem cells), it can be evaluated that the pluripotency of the mammalian cells (pluripotent stem cells) transferred onto the chorioallantoic membrane of a developing chicken egg is higher than the pluripotency of the comparative mammalian cells (pluripotent stem cells). Conversely, if teratoma formation is not observed in the above step (b), it can be evaluated that the mammalian cells transferred onto the chorioallantoic membrane of a developing chicken egg are not pluripotent stem cells (or the mammalian cell population transferred onto the chorioallantoic membrane of a developing chicken egg does not contain pluripotent stem cells). Furthermore, in the above step (b), if the teratoma formation rate of the mammalian cells (pluripotent stem cells) transferred onto the chorioallantoic membrane of a developing chicken egg is equal to or lower than the teratoma formation rate of the control mammalian cells (pluripotent stem cells), the pluripotency of the mammalian cells (pluripotent stem cells) transferred onto the chorioallantoic membrane of a developing chicken egg can be evaluated as being equal to or lower than the pluripotency of the control mammalian cells (pluripotent stem cells). Even if the mammalian cells transferred onto the chorioallantoic membrane of a developing chicken egg are multipotent stem cells or unipotent stem cells, which are included in the same concept of "stem cells" as pluripotent stem cells, it is considered that teratomas will not be formed in the above step (b) as long as they do not have pluripotency.

[0027] The present invention will be described in more detail below with reference to examples, but the technical scope of the present invention is not limited to these examples. EXAMPLES

[0028] 1. The present chorioallantoic epiteratoma formation method A slit was made in the shell of an embryonated chicken egg (obtained from Fukagawa Poultry Agricultural Cooperative) on the 10th day after the start of incubation, and 1–10 × 10 50.1 mL of PBS containing a mouse ES cell line (established from mice obtained from Japan SLC) at 10 cells / mL was injected onto the chorioallantoic membrane using a syringe. Developed chicken eggs into which a mouse ES cell line had been injected onto the chorioallantoic membrane were cultured at 37°C using an incubator and cracked 6 days after injection. Of the total number of eggs ("total" in Table 1), the number of infertile eggs, the number of eggs in which degeneration was observed ("degeneration" in Table 1), the number of eggs in which teratoma formation was not observed ("-" in Table 1), and the number of eggs in which teratoma formation was observed ("+" in Table 1) were measured, and the egg survival rate (= ("number of all eggs" - "number of eggs in which degeneration was observed) / "number of all eggs"), the proportion of eggs in which teratoma formation was observed to all eggs ("formation rate 1" in Table 1 = "number of eggs in which teratoma formation was observed" / "total number of eggs") ("formation rate 1" in Table 1), and the proportion of eggs in which teratoma formation was observed to all surviving eggs ("formation rate 2" in Table 1 = "number of eggs in which teratoma formation was observed" / ("total number of eggs" - "number of eggs in which degeneration was observed")) were calculated.

[0029] [Table 1] "2n_1" to "2n_4" indicate diploid mouse ES cell lines, "4n_1" to "4n_4" indicate tetraploid mouse ES cell lines, and "8n" indicates an octaploid mouse ES cell line.

[0030] 2.Results When the present chorioallantoic membrane teratoma formation method was performed using diploid mouse ES cell lines ("2n_1" to "2n_4"), which are conventional mouse ES cell lines, the egg survival rate was 70 to 90% (see "Egg survival rate (%)" in Table 1), and the teratoma formation rate ("the proportion of eggs in which teratoma formation was observed compared to the number of surviving eggs") was 55.6 to 68.8% (see "Formation rate 2" in Table 1).

[0031] On the other hand, when mouse ES cell lines were microinjected into developing chicken eggs using the method described in Non-Patent Document 1, the egg survival rate was 53.1 to 96% ("Surviving / Injected" in Table 1), and the teratoma formation rate (the percentage of eggs in which teratoma formation was observed compared to surviving eggs) was 34.9 to 82.4% (see "LacZ positive / Surviving" in Table 1).

[0032] These results show that the use of the present chorioallantoic membrane teratoma formation method can suppress the decrease in egg survival rate and the decrease in teratoma formation compared to the use of the method described in Non-Patent Document 1, and also makes it possible to perform teratoma formation with good reproducibility.

[0033] In addition, it is known that tetraploid mouse ES cell lines have a low teratoma formation ability based on a teratoma formation test using immunodeficient mice (see the literature "PLoS One. 2015 Jun 19;10(6):e0130585."). When the present chorioallantoic membrane teratoma formation method was performed using tetraploid mouse ES cell lines ("4n_1" to "4n_4"), the two types of teratoma formation rates ("the percentage of eggs in which teratoma formation was observed among all eggs" and "the percentage of eggs in which teratoma formation was observed among surviving eggs") were 25-45% and 29.4-56.3%, respectively, which were lower than the two types of teratoma formation rates of 45-55% and 55.6-68.8% when a diploid mouse ES cell line, which is a normal mouse ES cell line, was used (see Table 1). This result indicates that the results of the teratoma formation test using immunodeficient mice can be reproduced by the present chorioallantoic membrane teratoma formation method.

[0034] Furthermore, when the present chorioallantoic membrane teratoma formation method was performed using an octaploid mouse ES cell line ("8n"), which is expected to have a lower teratoma formation ability than a tetraploid mouse ES cell line, the two types of teratoma formation rates ("the percentage of eggs in which teratoma formation was observed out of all eggs" and "the percentage of eggs in which teratoma formation was observed out of surviving eggs") were 17% and 20%, respectively, which were shown to be even lower than the two types of teratoma formation rates of 25-45% and 29.4-56.3% when a tetraploid mouse ES cell line was used (see Table 1). These results indicate that by performing the present chorioallantoic membrane teratoma formation method and measuring the teratoma formation rate, it is possible to evaluate the level of teratoma-forming ability (i.e., pluripotency) of mammalian cells transferred onto the chorioallantoic membrane of developed chicken eggs. [Industrial Applicability]

[0035] The present invention contributes to the quality evaluation (multipotency evaluation) of pluripotent stem cells used in regenerative medicine and the like.

Claims

1. A method for assessing the pluripotency of a mammalian cell, comprising the steps of: (a) determining whether the cell is pluripotent; (a) transferring mammalian cells onto the chorioallantoic membrane of an embryonated chicken egg and then culturing the embryonated chicken egg; (b) evaluating the pluripotency of the mammalian cells using teratoma formation in the embryonated chicken eggs cultured in step (a) as an indicator;

2. The method according to claim 1, wherein in step (a), the mammalian cells are transferred onto the chorioallantoic membrane of an embryonated chicken egg on or after the 10th day after the start of incubation.

3. 3. The method according to claim 1 or 2, wherein in step (a), the embryonated chicken eggs are cultured for 6 days or more.

4. 3. The method according to claim 1 or 2, wherein in step (a), the mammalian cells are injected onto the chorioallantoic membrane of an embryonated chicken egg using a syringe.

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