How to Break Down Embryonic Membranes

Abstract

To provide a novel method for decomposing an embryonic membrane.MEANS FOR SOLVING THE PROBLEM: An embryonic membrane derived from a fertile egg of a bird or a reptile is treated with an enzymatic mixture comprising collagenase and / or papain, hyaluronidase, and DNaseI.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for degrading embryonic membranes. [Background technology]

[0002] Methods for culturing cells derived from animal embryonic membranes include culturing cells dispersed by enzymatically decomposing the embryonic membrane, culturing cell masses by physically disrupting the embryonic membrane, and culturing the embryonic membrane as tissue. When recovering cells from the embryonic membranes of birds or reptiles, the embryonic membranes must be decomposed to disperse the cells. Until now, enzymes known for decomposing embryonic membranes include trypsin and collagenase (see Non-Patent Documents 1 and 2). However, in conventional cell recovery methods, the stickiness of the embryonic membranes makes it time-consuming to pass the cells through a cell strainer, preventing efficient recovery. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Lin HJ et al., J Vis Exp. 2016 Mar 10;(109):53624. [Non-patent document 2] Wang SH et al., PLoS One. 2017 Nov 21;12(11):e0187560. Summary of the Invention [Problem to be solved by the invention]

[0004] An object of the present invention is to provide a method for efficiently decomposing embryonic membranes. [Means for solving the problem]

[0005] One embodiment of the present invention is a method for decomposing embryonic membranes derived from fertilized eggs of birds or reptiles, using an enzyme mixture containing collagenase and / or papain, hyaluronidase, and DNase I. The collagenase concentration may be 2.5 U / mL to 2500 U / mL or 25 U / mL to 2500 U / mL. The papain concentration may be 0.1 U / mL to 100 U / mL or 0.1 U / mL to 10 U / mL. The hyaluronidase concentration may be 1.5 U / mL to 1500 U / mL or 15 U / mL to 1500 U / mL. The DNase I concentration may be 5000 U / mL or less, or 50 U / mL to 5000 U / mL.

[0006] Another embodiment of the present invention is an enzyme mixture comprising collagenase and / or papain, hyaluronidase, and DNase I. The enzyme may be for degrading embryonic membranes, which may be derived from fertilized eggs of birds or reptiles.

[0007] A further embodiment of the present invention is a method for treating a fibroblast with collagenase and / or papain in combination with hyaluronidase.

[0008] The kit may comprise DNase and DNase I. The kit may be for decomposing embryonic membranes. The embryonic membranes may be derived from fertilized eggs of birds or reptiles. [Effects of the Invention]

[0009] The present invention provides an efficient method for decomposing embryonic membranes. [Brief explanation of the drawings]

[0010] [Figure 1] 1 is a graph showing the time required for avian embryonic membranes to pass through a cell strainer when treated with a triple enzyme mixture of collagenase, hyaluronidase, and DNase I in one example of the present invention. [Figure 2]1 is a graph showing the amount of tissue recovered when avian embryonic membranes were treated with a triple enzyme mixture of collagenase, hyaluronidase, and DNase I in one example of the present invention. [Figure 3] 1 is a graph showing the number of cells cultured after enzyme treatment when avian embryonic membranes were treated with a triple enzyme mixture of collagenase, hyaluronidase, and DNase I in one example of the present invention. [Figure 4] 1 is a graph comparing the cell strainer passage time in one example of the present invention, with a constant collagenase concentration and varying hyaluronidase concentrations, in the presence or absence of DNase I. [Figure 5] 1 is a graph showing a comparison of the cell strainer passage time in one example of the present invention, in which the hyaluronidase concentration was kept constant and the collagenase concentration was varied, with and without DNase I. [Figure 6] 1 is a graph comparing the cell strainer passage time when the collagenase concentration and hyaluronidase concentration are kept constant and the DNase I concentration is changed in one example of the present invention. [Figure 7] 1 is a graph showing the cell strainer passage time when a triple enzyme mixture containing any one of collagenases I to IV is used in one example of the present invention. [Figure 8] 1 is a graph comparing the cell strainer passage time in one example of the present invention, with a constant hyaluronidase concentration, varying papain concentrations, and the presence or absence of DNase I. [Figure 9] 1 is a graph showing the cell strainer passage time of a cell dispersion obtained by treating avian embryonic membranes with a triple enzyme mixture of trypsin, hyaluronidase, and DNase I in one example of the present invention. [Figure 10] 1 is a graph showing the number of cells in a cell dispersion obtained by treating avian embryonic membranes with a triple enzyme mixture of trypsin, hyaluronidase, and DNase I, during culture after enzyme treatment in one example of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0011] The objectives, features, advantages, and ideas of the present invention will be apparent to those skilled in the art from the description in this specification, and those skilled in the art will be able to easily reproduce the present invention. The embodiments and specific examples of the invention described below show preferred embodiments of the present invention and are shown for illustrative or explanatory purposes, and do not limit the present invention thereto. It will be apparent to those skilled in the art that various changes and modifications can be made based on the description in this specification within the spirit and scope of the present invention disclosed in this specification. <Method for decomposing embryonic membranes>

[0012] One embodiment of the present invention is a method for decomposing embryonic membranes, comprising the step of treating embryonic membranes derived from fertilized eggs of birds or reptiles with an enzyme mixture comprising collagenase (EC 3.4.24.3) (EC 3.4.24.7) and / or papain (EC 3.4.22.2), hyaluronidase (EC 3.2.1.35), and DNase I.

[0013] The embryonic membrane may be derived from a fertilized egg of a bird or reptile, and the type of bird or reptile is not particularly limited. The number of days after fertilization of the fertilized egg is not particularly limited, but for example, when collecting the embryonic membrane from a chicken, the period from day 1 to day 20 after fertilization is preferred, and the period from day 10 to day 15 is more preferred.

[0014] The concentration of collagenase is not particularly limited, but is preferably 2.5 U / mL or more and 2500 U / mL or less, and more preferably 25 U / mL or more and 2500 U / mL or less.

[0015] The collagenase may be crude collagenase or purified collagenase purified from animal cells or bacteria, or recombinant collagenase extracted from animal cells, yeast, or bacteria after expressing a recombinant gene in those cells, or commercially available collagenase may be used, with no particular limitation on the product.

[0016] The type of collagenase is not particularly limited, and may be one or more enzymes selected from the group consisting of collagenases I to VII.

[0017] The concentration of papain is not particularly limited, but is preferably 0.1 U / mL to 100 U / mL, more preferably 0.1 U / mL to 10 U / mL.

[0018] Papain may be crude or purified papain purified from plants, or recombinant papain extracted from animal cells, yeast, or bacteria after expressing a recombinant gene in those cells.

[0019] The concentration of hyaluronidase is not particularly limited, but is preferably 1.5 U / mL or more and 1500 U / mL or less, and more preferably 15 U / mL or more and 1500 U / mL or less.

[0020] The hyaluronidase may be crude hyaluronidase or purified hyaluronidase purified from animals, or it may be recombinant hyaluronidase extracted from animal cells, yeast, or bacteria after expressing a recombinant gene in those cells.

[0021] DNase I is an enzyme that catalyzes the degradation of single-stranded and double-stranded DNA to produce mononucleotides or oligonucleotides containing a 5' phosphate group. DNase I may be purified from natural sources or obtained by expressing a recombinant gene in a host cell. For example, DNase I may be obtained by expressing a bovine pancreatic DNase I gene in yeast. RNase-free DNase I is preferred.

[0022] The concentration of DNase I is not particularly limited, but is preferably more than 0 U / mL and not more than 5000 U / mL, and more preferably not less than 50 U / mL and not more than 5000 U / mL.

[0023] DNase I may be crudely purified DNase I or purified DNase I purified from animals, or recombinant DNase I extracted from animal cells, yeast, or bacteria after expressing a recombinant gene in those cells.

[0024] The mixed enzymes contain these three or four enzymes at predetermined concentrations.

[0025] Specifically, the embryonic membrane is first extracted from fertilized eggs of birds or reptiles, cut appropriately, washed, and then treated with a mixed enzyme. After dispersing the cells by pipetting or the like, cell clumps and debris are removed by passing through a cell strainer or the like. By using this method, the embryonic membrane is disassembled, and embryonic membrane-derived cells can be obtained in a dispersed state.

[0026] <Kit> The kit of one embodiment of the present invention comprises collagenase and / or papain, hyaluronidase, and DNase I. This kit can be used to decompose embryonic membranes.

[0027] When the kit contains collagenase, the ratio of the activity units of the enzymes contained in the kit, i.e., collagenase:hyaluronidase:DNase I, is preferably 2.5-2500:1.5-1500:0.1-5000, and more preferably 25-2500:15-1500:5-5000. When the kit contains papain, the ratio of the activity units of the enzymes contained in the kit, i.e., papain:hyaluronidase:DNase I, is preferably 0.1-100:1.5-1500:0.1-5000, and more preferably 0.1-10:15-1500:50-5000. When both collagenase and papain are contained, the ratio of enzyme activity units is preferably collagenase:papain:hyaluronidase:DNase I of 2.5-2500:0.1-100:1.5-1500:0.1-5000, more preferably 25-2500:0.1-10:15-1500:5-5000. [Example]

[0028] <Method> (1) Collection of embryonic membranes

[0029] Embryonic membranes were collected from fertilized chicken eggs 13 days after incubation and finely sheared in HBSS. (2) Decomposition of embryonic membranes

[0030] The sheared embryonic membranes were washed with HBSS, immersed in the enzyme solution, and incubated for 1 hour at 37°C. After that, the cells were dispersed by pipetting, and the cell dispersion was processed with a cell strainer with a mesh size of 100 μm.

[0031] (3)Cell culture The number of viable cells in the cell suspension that passed through the cell strainer was counted. The obtained cells were plated in a 12-well plate at 45,000 cells / cm. 2 The seeds were sown at a density of .

[0032] The cells were cultured in DMEM containing 10% FBS and 1% Penicillin-Streptomycin-Amphotericin B Suspension at 37°C in the presence of 5% CO2.

[0033] (4)Measurement method (4-1) Cell strainer passage time

[0034] Video was taken while the cell dispersion was being processed through the cell strainer. The frame rate was fixed at 30 fps, and the number of frames from when the cell dispersion was added until all of the cells had passed through was measured, and this was taken as the cell strainer passage time. In this measurement, it is believed that the greater the degree of cell dispersion, the shorter the cell strainer passage time.

[0035] (4-2) Amount of tissue recovered The amount of tissue recovered was calculated by subtracting the weight of the treated embryonic membrane from the residue on the cell strainer. It is believed that the greater the degree of tissue dispersion, the fewer cell masses will remain on the cell strainer, and the greater the amount of tissue recovered.

[0036] (4-3) Cell count in culture after enzyme treatment The cells were cultured after enzyme treatment, and 2, 4, and 8 days after the start of culture, the cell nuclei were stained with Hoechst 33342 and images of the stained nuclei were taken using a fluorescence microscope. The nuclear signals in the images were counted using image analysis software, and the total cell number was calculated. This allowed us to determine the proliferation of the cells after enzyme treatment, and if the cells were healthy, cell proliferation would be observed within 2 to 8 days.

[0037] <Experimental conditions and results> (1) Comparison with single enzymes

[0038] Approximately 1 g of embryonic membranes was treated with the enzymes or triple enzyme mixtures shown in Table 1. Using the methods described above, the time it took to pass through the cell strainer, the amount of tissue recovered, and the number of cells in culture after enzyme treatment were measured. The results obtained are shown in Figures 1 to 3, with the results for treatment with collagenase II alone set at 100%. [Table 1]

[0039] As a result, the cell suspension passed through the cell strainer the fastest with the triple enzyme mixture (Figure 1), and the amount of tissue recovered was also the greatest with the triple enzyme mixture (Figure 2).Furthermore, while fewer adherent cells were recovered with hyaluronidase or DNase I alone than with collagenase II alone, the triple enzyme mixture yielded approximately the same number of adherent and proliferative cells as collagenase II alone (Figure 3).

[0040] (2) Comparison with a two-type mixed enzyme Approximately 1 g of embryonic membranes was treated with the enzyme mixtures shown in Tables 2 and 3. The time required for passage through the cell strainer was measured using the method described above, and the results are shown in Figures 4 and 5, with the results for treatment with collagenase II alone (Table 2) and hyaluronidase alone (Table 3), respectively, set at 100%. [Table 2] [Table 3]

[0041] As a result, the triple enzyme mixture with DNase I added showed a shorter cell strainer passage time, even when the concentrations of the other two enzymes were changed (Figures 4 and 5). Furthermore, a tendency for the cell strainer passage time to become shorter depending on the collagenase II and hyaluronidase concentrations was observed (Figures 4 and 5).

[0042] (3) DNase I concentration dependence As shown in Table 4, collagenase II and hyaluronidase were used at constant concentrations, and DNase I was used at five concentrations between 0 and 5000 U / mL, and the time it took to pass through the cell strainer was measured. The results obtained when treatment was performed without adding DNase I were set to 100%, and are shown in Figure 6. [Table 4]

[0043] As a result, under the experimental conditions, 50 U / mL of DNase I showed almost the maximum effect on the cell strainer passage time.

[0044] (4) Types of collagenase As shown in Table 5, the cell strainer passage time was measured when a triple enzyme mixture containing collagenases I to IV was used, and the results obtained when treated with collagenase II alone were set to 100%, and are shown in Figure 7. [Table 5]

[0045] As a result, regardless of which collagenase was used, a significant reduction in the cell strainer passage time was observed compared to when collagenase II alone was used.

[0046] (5) Use of papain As shown in Table 6, a triple enzyme mixture containing papain was used instead of collagenase, and the cell strainer passage time was measured at multiple concentrations. The results obtained are shown in Figure 8, with the result for treatment with hyaluronidase alone set at 100%. [Table 6]

[0047] As a result, even when papain was used, the time required for passage through the cell strainer was shortened, as was the case with collagenase.

[0048] (6) Use of trypsin As shown in Table 7, a triple enzyme mixture containing trypsin instead of collagenase was used, and the cell strainer passage time was measured at multiple concentrations. The results were shown in Figure 9, with the result for Sample 1 set to 100%. In addition, the number of cells in culture after enzyme treatment was measured, and the results were shown in Figure 10, with the result for Sample 6 set to 100%. [Table 7]

[0049] As a result, there was no difference in the time it took for cells to pass through the cell strainer when trypsin was used compared to when it was not used. Furthermore, the enzyme-treated cells did not proliferate when cultured. This is thought to be because trypsin is a strong proteolytic enzyme, and the cells are damaged by trypsin.

[0050] (8) Summary As shown in the above examples, when embryonic membranes derived from fertilized eggs of birds or reptiles are treated with an enzyme mixture containing collagenase and / or papain, hyaluronidase, and DNase I, the time required for the cells to pass through a cell strainer is significantly reduced and the degree of cell dispersion increases, making it possible to recover culturable cells with minimal enzyme damage.

Claims

1. A method for decomposing embryonic membranes, comprising a step of treating embryonic membranes derived from fertilized avian eggs with a mixed enzyme comprising collagenase and / or papain, hyaluronidase, and DNase I.

2. The method for decomposing embryonic membranes according to claim 1, wherein the concentration of the collagenase is 2.5 U / mL or more and 2500 U / mL or less.

3. The method for decomposing embryonic membranes according to claim 2, wherein the concentration of the collagenase is 25 U / mL or more and 2500 U / mL or less.

4. 2. The method for decomposing embryonic membranes according to claim 1, wherein the concentration of papain is 0.1 U / mL or more and 100 U / mL or less.

5. The method for decomposing embryonic membranes according to claim 4, wherein the concentration of papain is 0.1 U / mL or more and 10 U / mL or less.

6. The method for decomposing embryonic membranes according to any one of claims 1 to 5, wherein the concentration of the hyaluronidase is 1.5 U / mL or more and 1500 U / mL or less.

7. The method for decomposing embryonic membranes according to claim 6, wherein the concentration of the hyaluronidase is 15 U / mL or more and 1500 U / mL or less.

8. The method for decomposing embryonic membranes according to any one of claims 1 to 7, wherein the concentration of the DNase I is 5000 U / mL or less.

9. The method for decomposing embryonic membranes according to claim 8, wherein the concentration of DNase I is 50 U / mL or more and 5000 U / mL or less.

10. An enzyme mixture for decomposing embryonic membranes derived from fertilized avian eggs, comprising: An enzyme mixture comprising collagenase and / or papain, hyaluronidase, and DNase I.

11. A kit for decomposing embryonic membranes derived from fertilized avian eggs, comprising: A kit comprising collagenase and / or papain, hyaluronidase, and DNase I.

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