KR101794544B1 - METHOD FOR IMPROVING BrdU ASSAY - Google Patents

Abstract

The present invention relates to a method for improving bromodeoxyuridine (BrdU) assay which is one of cell proliferation assay. More specifically, provided is a method to use 4MBr-5 cells in a BrdU assay right after defrosting regardless of storage periodes to preserve the 4MBr-5 cells by freezing, without requiring long-term incubation. Accordingly, it is possible to easily and quickly measure biological activities of protein involved in cell proliferation such as FGF7 through the BrdU assay.

Description

[0001] METHOD FOR IMPROVING BrdU ASSAY [0002]

The present invention relates to a method for improving the BrdU assay, one of the cell proliferation assays.

More specifically, the present invention provides a method which can be used immediately for BrdU assay without long term incubation after thawing regardless of the storage period of 4MBr-5 cells. Accordingly, it is possible by the present invention to more quickly and easily measure the biological activity of proteins involved in cell proliferation such as FGF7 through the BrdU assay.

FGF7 (Fibroblast Growth Factor 7) is a protein belonging to the FGF family, which is also known as KGF (Keratinocyte growth factor). FGF7 protein is a growth factor that functions when it is secreted mainly from epithelial cells. Major functions include development, morphogenesis, angiogenesis, wound healing, tumorigenesis, And tumor growth.

In particular, it has recently been demonstrated that FGF7 protein is effective in healing wounds by promoting the proliferation of epithelial cells and epidermal cells. Accordingly, in the field of the art, not only the commercially available FGF7 protein is purchased and studied, but efforts have been made to produce FGF7 recombinant protein having better biological activity by using its own transformation technique or the like.

At this time, it is necessary to confirm whether the newly produced FGF7 recombinant protein normally exhibits the biological activity. If the self-produced FGF7 recombinant protein does not exhibit normal biological activity, such FGF7 recombinant protein can not be suitably used.

Therefore, it is necessary to confirm the biological activity of the FGF7 protein in vitro in advance. As one of the widely used cell proliferation assays, a BrdU assay (Bromodeoxyuridine, 5-bromo-2 ' -deoxyuridine assay).

Conventionally, in order to confirm and measure the degree of cell proliferation, a method of measuring the degree of cell proliferation by radioactive labeling using isotope 3H-thymidine when DNA is synthesized during cell division has been used. However, due to the risks associated with the use of the radioactive isotope 3H-thymidine, a BrdU assay was developed to replace this.

The BrdU assay is based on the principle of measuring cell proliferation through a colorimetric method in which labeled U is incorporated into newly synthesized DNA at the time of cell division and immunodominated using a BrdU-specific antibody . For example, when FGF7 is added to a medium for culturing epithelial cells, epithelial cells are proliferated. By measuring the amount of newly synthesized DNA by staining with a BrdU-specific antibody, the presence or absence of cell proliferation activity of FGF7 Can be identified and measured.

4MBr-5 cells (rhesus monkey epithelial cells), BaF3, Balb / mk cells, etc. are mainly used as the types of cells treated with the FGF7 protein to measure the activity of the FGF7 protein in the BrdU assay.

When performing the BrdU assay, 4MBr-5 cells are cultured for several generations and used whenever necessary. However, in this case, there is a problem that 4MBr-5 cells are exposed to mycoplasma, virus, etc. during the subculture, and the subcultured 4MBr-5 cells are more likely to be infected with each other, And so on, are variables that hinder the uniformity of the BrdU assay results.

For these reasons, there is a method of mass-producing 4MBr-5 cells in a large amount, freezing and preserving them, and using them when needed, rather than using 4MBr-5 cells in subculture at the time of performing the BrdU assay Is preferred.

To freeze adherent cells such as 4MBr-5 cells, a freezing stock solution was prepared by adding 7.5% to 10% DMSO (dimethyl sulfoxide: dimethyl sulfoxide) to the medium used for culturing the cells, It is customary to store the cells in a liquid tube with a Deepfreezer at -70 ° C after adding them to a storage tube or vial with this frozen stock solution.

However, since the above frozen stock solution is basically intended for long-term storage of cells, various problems arise when it is ultimately aimed to perform BrdU assay after thawing 4MBr-5 cells.

For example, since 4MBr-5 cells are frequently damaged during freezing and thawing, they must undergo a stabilizing process to restore them after thawing. 4MBr-5 cells are in normal exponential growth phase (log phase) It takes a short 7 days to reach and a month or longer. In particular, since 4MBr-5 cells are slow to grow, it takes a long time to thaw the frozen cells and reach the stabilization step.

If the cell stabilization takes a long period of time after such thawing, there is a high possibility that the cells are exposed to various kinds of contamination in the same manner as the method of using the cells without subculturing. In addition, when cells were frozen at different time points for use in the BrdU assay during storage of frozen 4MBr-5 cells, the 4MBr-5 cells showed significant deviations in the time to reach the exponential phase (log phase) And thus the cell growth and / or proliferation rate, protein expression ability, etc. are different from each other, resulting in a significant error in the results of the BrdU assay and significantly lowering the reliability. The large deviation of the experimental result and the lowering of the reliability also bring about a problem that a person skilled in the art has to perform more repeated experiments.

In addition, cells that have been stored frozen with the frozen stock solution in tubes or vials need to be pipetted onto well-plates to perform the BrdU assay after thawing. However, this process causes damage to the cells again after thawing, and there is also a need to separately ascertain whether the thawed cells adhere to the bottom of the transferred plate and grow.

Thus, when 4MBr-5 cells that were kept frozen were used for BrdU assay, the 4MBr-5 cells were transferred from the tube or vial to the well-plate and / or the 4MBr-5 cells were stabilized for a prolonged period If the process can be omitted or reduced, the accuracy and reliability of the BrdU assay will be significantly increased and eventually the FGF7 protein activity will be more accurately and easily measured.

The inventors of the present invention conducted various experiments in order to solve the problems of the prior art described above. As a result, whether or not 4MBr-5 cells stored in a freeze-thawed state can be immediately used for BrdU assay, And the number of cells and the type of freezing stock solution used.

The method according to the present invention is an improvement of a method for performing a BrdU assay known in the art for measuring the epithelial cell or epidermal cell proliferative activity of FGF7 protein, and comprises the following steps.

a) freezing epithelial cells or epidermal cells with a freezing stock solution consisting of 10% DMSO, 10% FBS and 80% F-12K medium, and storing until the BrdU assay is performed;

b) defrosting epithelial cells or epidermal cells that are frozen in step a); And

c) carrying out a BrdU assay by adding FGF7 protein to epithelial cells or epidermal cells that have been thawed in step b).

In a preferred embodiment, the method according to the invention comprises the following steps:

a-0) epithelial cells or epidermal cells in F-12K medium supplemented with 10% FBS;

a) freezing epithelial cells or epidermal cells with a freezing stock solution consisting of 10% DMSO, 10% FBS and 80% F-12K medium, and storing until the BrdU assay is performed;

b) defrosting epithelial cells or epidermal cells that are frozen in step a); And

c) carrying out a BrdU assay by adding FGF7 protein to epithelial cells or epidermal cells that have been thawed in step b).

In a preferred embodiment, the method according to the invention comprises the following steps:

a-0) epithelial cells or epidermal cells in F-12K medium supplemented with 10% FBS;

a) freezing epithelial cells or epidermal cells with a freezing stock solution consisting of 10% DMSO, 10% FBS and 80% F-12K medium, and storing until the BrdU assay is performed;

b) defrosting epithelial cells or epidermal cells that are frozen in step a);

c-0) culturing in an F-12K medium supplemented with 2% FBS without washing the thawed epithelial cells or epidermal cells; And

c) carrying out a BrdU assay by adding FGF7 protein to epithelial cells or epidermal cells that have been thawed in step b).

The cells frozen in step a) may be stored for a desired period of time. For example, for one to eight weeks or more.

It is preferable that the period for culturing the cells thawed in the step c-0) is performed within as short a time as possible. It is more preferred to be carried out for one to two days, in particular for one day.

The cells are characterized in that the cells are contained in a multi-well plate in both a-0), a), b), c-0) and c). That is, in step a-0), the cells are cultured in a state contained in a multi-well plate. In step a), the cells are frozen and stored as contained in a multi-well plate. In step b) The cells are cultured in a state contained in the multi-well plate in step c-0), and in step c), the cells are added to the multi-well plate and the FGF7 protein is added BrdU assay is performed.

The multi-well plate can be, but is not limited to, a 6-well, 12-well, 24-well, 48-well or 96-well plate. It is more preferable that the multi-well plate is a 96-well plate, since the absorbance can be directly measured when using a 96-well plate, which is advantageous in terms of convenience and time.

If the multi-well plate is a 96-well plate, it is preferable that ⁴ MBr-5 cells are dispensed at a concentration of ⁴ × 10 ⁴ to 6 × 10 ⁴ cells / well per well. Particularly, it is more preferable to dispense cells at a concentration of ⁵ x 10 ⁴ cells (0.2 ml each at a concentration of 2.5 x 10 ⁵ cells / ml per well).

The FGF7 protein may be a hFGF7 recombinant protein produced directly by the present applicant.

The epithelial cells or epidermal cells may be 4MBr-5 cells, but are not limited thereto, and include all kinds of cells in which the FGF7 protein can exhibit cell proliferation activity.

The medium may further comprise EGF at a concentration of, for example, 30 ng / ml.

When the method of freezing NB324K human kidney cells, HaCaT human keratinocyte cells, RH18 human muscle cells and the like is applied to 4MBr-5 cells as well, there is no problem in freezing 4MBr-5 cells and storing them for a long period of time.

However, in the case of using BrdU assay to measure the activity of FGF7 recombinant protein in a short time after defrosting 4MBr-5 cells, conventional freezing conditions are not suitable.

For example, tubes or vials are typically used for convenient storage, but there is a procedural hurdle where the 4MBr-5 cells must be thawed and then dispensed to the correct number of cells on the well-plate. In addition, cells are often damaged during the freezing and thawing process, resulting in further damage to 4MBr-5 cells through the process of transferring cells from a tube or vial to a well-plate.

Furthermore, even after 4 MBr-5 cells are thawed and dispensed on well-plates, the cells should be well adhered to the bottom of the plate and well grown to the normal exponential phase (log phase) To more than one month, and it is difficult to predict the exact period of time it takes. In particular, 4MBr-5 cells are slow growing and do not grow well, so they tend to grow much slower if the conditions involved in nutrient and cell growth are insufficient, resulting in a time-consuming problem before starting the BrdU assay do.

In addition, when the 4MBr-5 cells are cultured and the period of reaching the log phase is prolonged, there is a large variation in cell growth rate, survival rate, and proliferation rate in each cell group. As a result, when the BrdU assay is performed using 4MBr-5 cells, there is a large error in the experimental results, and more repeated experiments are required to increase the reliability of the experimental results.

On the other hand, according to the method of the present invention, a large amount of 4MBr-5 cells having homogeneous properties such as growth rate, survival rate, proliferation rate and the like of the cells are cultured in large quantities, and then the BrdU assay is performed on a well plate 4MBr-5 cells are pre-divided and frozen. When the biological activity of the newly prepared FGF7 recombinant protein is to be measured, a well plate containing 4 MBr-5 cells can be thawed and used for the BrdU assay.

Therefore, the present invention does not require a process of transferring a 4MBr-5 cell from a tube or vial to a well-plate when the 4MBr-5 cells are thawed and cultured to perform the BrdU assay, thereby reducing the time and effort And is particularly advantageous in that it can eliminate the process of damaging cells.

Since the 4MBr-5 cells having homogeneous properties are stored in the frozen well plate, for example, 4MBr-5 cells used for thawing after 1 week and thawed after 8 weeks or more are used 4MBr-5 cells are almost identical in nature, so that the parameters affecting the results of the BrdU assay can be significantly reduced. This means that it is possible to thaw and use 4MBr-5 cells that are kept frozen at any time when the BrdU assay needs to be performed.

In particular, as demonstrated in the experiments in the Examples below, when the composition of the frozen stock solution according to the present invention is used to freeze 4MBr-5 cells, the BrdU assay is performed without freezing 4MBr-5 cells The results of the BrdU assay can be obtained to the same extent as the results obtained. The reason for this is that since the period of reaching the exponential phase is very short after 4 MBr-5 cells are thawed and cultured, the characteristics such as growth rate and cell proliferation rate are almost different when compared with 4 MBr-5 cells before freezing It is thought that it is because it does not. This means that even if 4MBr-5 cells are stored frozen, the activity of FGF7 protein can be measured with high reliability and accuracy, just as it is not frozen.

In addition, the possibility that 4MBr-5 cells are infected by exposure to mycoplasma, virus, etc. can be minimized.

FIG. 1 is a micrograph taken on day 2 after treatment of FMB7 recombinant protein after starvation in two groups of w / o FBS and 2% FBS for non-frozen 4MBr-5 cells. Two representative photographs of each of several well photographs are shown.
After 2 is diluted 4MBr-5 cells to ^{4x10 4 cells / well, 6x10 4} cells / well, 12x10 4 cells / well ( each ^{2x10 5 cells / ml, 3x10 5} cells / ml, 6x10 5 cells / ml concentration of (Final concentration per well after 0.2 ml per well) was cultured in a 96-well plate, and then photographed by a microscope before freezing and then observed on the first day after thawing after freezing. A representative photograph is shown among several well photographs.
3 is a photomicrograph of the 4MBr-5 cells according to the first to fourth protocol under the conditions of the A and B experimental groups, followed by observation on the first day after thawing and after starvation. Figure 3a, 3b and 3c is the cell number was carried out respectively by ^{4x10 4, 6x10 4, 12x10 4} cells / well. A representative photograph is shown among several well photographs.
FIG. 4 is a microscope photograph of the 4MBr-5 cells observed under the conditions of Experiment group 1 according to the first to fourth protocols. FIG. 4A, 4B and 4C show that 4MBr-5 cells were diluted to a concentration of 4x10 ⁴ , 5x10 ⁴ , 6x10 ⁴ cells / well (2x10 ⁵ cells / ml, 2.5x10 ⁵ cells / ml, 3x10 ⁵ cells / (Final concentration per well as a result of dispensing 0.2 ml per well), representative photographs are shown among several well photographs.
FIG. 5 is a graph showing the results of the first day of starvation in 2% FBS-containing medium without washing after 4MBr-5 cells which were stored for 1 week, 4 weeks and 8 weeks were frozen at 5 × 10 ⁴ cells / Lt; RTI ID = 0.0 > FGF7 < / RTI > A representative photograph is shown among several well photographs.
FIG. 6A shows the results of BrdU assay performed on rhFGF7 recombinant protein produced by Applicant and rhFGF7 protein commercially available from Speed Biosystems using 4MBr-5 cells without undergoing freezing and thawing As shown in the graph.
FIG. 6B is a graph showing the results of the BrdU assay performed on the rhFGF7 recombinant protein produced by the present applicant using 4MBr-5 cells which were stored and thawed for 8 weeks according to the present invention.

The present invention will be described in more detail based on the following examples, but it is not intended to limit the scope of the present invention. In addition, those skilled in the art will appreciate that various modifications and changes may be made thereto without departing from the spirit of the invention.

1. Preparation of experimental material

The materials used in the experiments of the present invention are as follows.

i. 4MBr-5 cells: ATCC CCL-208, rhesus monkey epithelial cells, showing the form of adherent growth

ii. rhFGF7 (recombinant human fibroblast growth factor 7):

(1) Directly produced by the present applicant through a transformation technique, obtained by expression in CHO cells

(2) purchased from Speed Biosystems as Cat # SDP1028 for use as a positive control, and obtained by expression in HEK 293 cells

iii. F-12K medium: GIBCO, Cat # 21127-022

The F-12K medium is a modified medium of Ham's F-12 Nutirent Mixture containing L-glutamine but not growth factor and FBS

iv. FBS (Fetal Bovine Serum): INVITROGEN, Cat # 10091-148, Lot # 8185628

vi. EGF (Epidermal Growth Factor): R & D, Cat # 2028-EG.

vii. BrdU cell proliferation assay kit: Cell signaling, Cat # 6813S

2. Determination of conditions to improve BrdU assay

a. Badge and starvation conditions

RPMI1640 medium has conventionally been used for the purpose of storing NB324K human kidney cells, HaCaT human keratinocyte cells, RH18 human muscle cells and the like by freezing. However, as described in the applicant's unpublished patent application No. 10-2015-0171086, the RPMI1640 medium was used to determine whether ATDC5 cells, which are mouse embryonic epithelial cells, undergo freezing and thawing during growth and proliferation (The growth and proliferation rates were significantly slower on the first and second days of culture, and viable cells were not adhered to the bottom surface of the plate on the third day of culture), and rhesus monkeys RPMI1640 medium was excluded from the base medium of 4MBr-5 epithelial cells. Experiments on a number of media indicated that F-12K medium was selected as the primary medium for 4MBr-5 cells used for the BrdU assay in the present invention.

It was then confirmed whether the FBS composition of the F-12K medium used in the starvation process was modified to affect subsequent BrdU assays.

The specific composition of the medium used in this experiment is shown in Table 1 below. When using%, this is based on volume.

Medium composition Cell maintenance medium F-12K medium + 10% FBS + 30ng / ml EGF starvation 1 F-12K medium + w / o FBS starvation 2 F-12K medium + 2% FBS

4MBr-5 cells were dispensed at a concentration of 2 x 10 ⁴ cells / well (0.2 ml / well of cells at a concentration of 1 × 10 ⁵ cells / ml) into a 96-well plate containing the cell maintenance medium described in Table 1 above. Then, the cells were cultured for 24 hours in culture at 37 ℃ incubator, 5% CO ₂ condition.

Then, 4MBr-5 cells starved with 2% FBS and w / o FBS media were treated with FGF7 recombinant protein, and the number and proliferation of cells were observed on the second day of culture (FIG. 1).

As shown in FIG. 1, the 4MBr-5 cells cultured in F-12K medium were cultured in starvation medium of w / o FBS and 2% FBS for 24 hours and then treated with FGF7 recombinant protein. starvation was faster than that of starvation in w / o FBS medium. w / o FBS, and 2% FBS medium did not show any significant difference in cell growth, but in the subsequent experiments procedures for performing starvation in 2% FBS medium were applied.

b. Cell concentration condition

It is an object of the present invention to improve the accuracy and speed of the BrdU assay when used in a BrdU assay after thawing 4MBr-5 cells stored frozen. Therefore, an experiment was conducted to find the concentration range of 4MBr-5 cells desirable for carrying out the BrdU assay.

This experiment was designed to find a condition that would shorten the period between the time at which 4MBr-5 cells were thawed and the time at which BrdU assay could be performed as short as possible. In particular, since 4MBr-5 cells are slow to grow and do not grow well, it is necessary to consider the time to reach a normal log phase so that the BrdU assay can proceed. Thus, we tried to find the optimal cell concentration in the well-plate just before freezing 4MBr-5 cells.

First, 4MBr-5 4x10 cells in 96-well plates ^{4 cells / well, 6x10 4 cells} / well, 12x10 4 cells / well ( each ^{2x10 5 cells / ml, 3x10 5} cells / ml, 6x10 5 cells / ml concentration of , And then 0.2 ml / well was added to each well at a final concentration per well), and the cells were cultured at 37 占 폚 for 1 day.

A frozen stock solution consisting of 10% DMSO, 10% FBS and 80% F-12K medium was prepared, and the 96-well plate containing 4MBr-5 cells was frozen at -70 ° C Deepfreezer together with the frozen stock solution. Seven days later, 4 MBr-5 cells were thawed, washed with PBS, and replaced with F-12K medium containing 2% FBS.

Fig. 2 shows a micrograph of the cell status observed on the first day of starvation before and after freezing according to the number of seeds of 4MBr-5 cells. As shown in FIG. 2, 4MBr-5 cells frozen in a frozen stock solution composed of 10% DMSO, 10% FBS and 80% F-12K medium according to the present invention were thawed, ⁴ × ^{10 4} cells / well, 6 × ^{10 4} cells / well, and 12 × 10 ⁴ cells / well.

However, when cell concentrations of 6x10 ⁴ cells / well and 12x10 ⁴ cells / well were applied, too much of the cells were grown in well-plates to the point where the BrdU assay could proceed. The method according to the present invention has significant advantages in that 4MBr-5 cells are not transferred to other vessels, such as tubes or vials, but continue to use the same well-plates. After the thawing of the frozen cells followed by the BrdU assay, If the plate is nearly saturated, it becomes difficult to determine how much FGF7 recombinant protein has proliferative activity of 4MBr-5 cells.

On the other hand, when the cell concentration of 4x10 ⁴ cells / well was applied, when freezing cells were thawed and then replaced with a medium containing 2% FBS, 4MBr-5 cells adhered to the bottom surface in a 96- Many wells in a floating state appeared. This leads to an error in the experiment as it causes a deviation between the wells.

Therefore, considering the above results of this experiment, in the subsequent experiment using the washing process as a variable, the concentration of 4 MBr-5 cells 4x10 ⁴ cells / well, 5x10 ⁴ cells / well, and 6x10 ⁴ cells / well (diluted to a concentration of 2x10 ⁵ cells / ml, 2.5x10 ⁵ cells / ml and 3x10 ⁵ cells / ml, The final concentration per well of the result).

c. The composition of the freezing stock solution

It is an object of the present invention to more accurately and quickly measure the cell proliferation activity of FGF7 recombinant protein in BrdU assay. Therefore, in order to determine the conditions for freezing and thawing 4MBr-5 cells, F-12K medium is set as a basic medium This experiment was designed by modifying the composition ratio of DMSO and FBS constituting the frozen stock solution.

First, 10% FBS the 4MBr-5 cells on the F-12K medium containing ^{4x10 4 cells / well, 6x10 4} cells / well, 12x10 4 cells / well ( each ^{2x10 5 cells / ml, 3x10 5} cells / ml, 6x10 ⁵ cells / ml, and 0.2 ml / well was diluted to a final concentration per well), and the solution was dispensed into each well of the plate. Thereafter, the cells were incubated in a 37 ° C incubator for 1 day, and the next day, 4 MBr-5 cells were attached to the bottom surface of the plate.

Next, as shown in Table 2 below, freezing stock solutions having different compositions of DMSO and FBS were prepared, and A and B experimental groups were constructed. The frozen stock solution was filled into each well of the plate by 200 mu l.

In the present invention, 4MBr-5 cells were frozen by storing the cells in a plate containing 4MBr-5 cells at -70 ° C in a Deepfreezer without transferring the cells to a container commonly used for storing cells such as tubes or vials. % Represents v / v.

Experimental group

DMSO
FBS
F-12K medium
Group A

10%
10%
80%
Group B

10%
20%
70%

Plates containing 4MBr-5 cells were frozen and stored for 4 weeks. Then, the well-plate containing 4 MBr-5 cells was thawed, and as shown in the following Table 3, the conditions of washing, stabilization and starvation were varied as in the first to fourth protocols A comparative experiment was performed.

After thawing
stabilize
After stabilization
Starvation
The first protocol
PBS wash
F-12K medium supplemented with 10% FBS
PBS or media wash
F-12K medium supplemented with 2% FBS

The second protocol
Do not wash
F-12K medium supplemented with 10% FBS

Do not wash
F-12K medium supplemented with 2% FBS


After thawing
Stabilization and starvation
Third protocol

PBS wash
F-12K medium supplemented with 2% FBS
Fourth protocol

Do not wash
F-12K medium supplemented with 2% FBS

After the washing step in Table 3 above, when F-12K medium containing 10% FBS or 2% FBS, respectively, was added to the plate, 200 μl / well was added and cultured.

In the first protocol and the second protocol of Table 3, after thawing, the cells were stabilized for one day with F-12K medium containing 10% FBS irrespective of washing, and then F-12K medium containing 2% FBS And then starvation process.

In the third protocol and the fourth protocol of Table 3, the F-12K medium containing 2% FBS was directly stabilized and starvated regardless of whether it was washed or not. On the second day after thawing, 4MBr-5 cells were well grown and observed whether they adhered well to the bottom of the plate.

FIG. 3 shows the results of the second protocol and the fourth protocol of Table 3 for the experimental group A and the experimental group B of Table 2, respectively.

FIG. 3 shows a case where the concentration of ⁴ MBr-5 cells is ⁴ × 10 ⁴ cells / well in F-12K medium containing 10% FBS, FIG. ³ cells / well, and FIG. 3C shows a case of 12x10 ⁴ cells / well.

As shown in the upper part of FIG. 3A, the A test group frozen with 4 MBr-5 cells in a frozen stock solution (% is a volume ratio) composed of 10% DMSO, 10% FBS and 80% F- It was observed that 4 MBr-5 cells grew well attached to the bottom surface of the plate. Then, when starvation was performed with F-12K medium containing 2% FBS, A-treated group was also grown with 4MBr-5 cells well attached to B-treated group.

On the other hand, as shown in the lower part of FIG. 3A, in the group B in which 4 MBr-5 cells were frozen in a frozen stock solution (% is a volume ratio) composed of 10% DMSO, 20% FBS and 70% F- On day 1, poor growth of 4MBr-5 cells was observed. Furthermore, as the starvation process proceeded, 4MBr-5 cells were observed not to adhere well to the bottom of the plate but to float separately.

In FIG. 3B and FIG. 3C in which the cell concentration is different from FIG. 3A, in the B test group, the 4MBr-5 cells were more prominent or more likely to grow or not adhere.

Therefore, it was found that a frozen stock solution composed of 10% DMSO, 10% FBS and 80% F-12K medium regardless of cell concentration was preferable to a frozen stock solution composed of 10% DMSO, 20% FBS and 70% F-12K medium Proven.

d. Stabilization process after thawing

In FIG. 3, when the second protocol and the fourth protocol are compared, it is possible to confirm the difference according to the presence or absence of the stabilization process after thawing.

The fourth protocol, in which the stabilization and starvation process were performed in a medium containing 2% FBS at once, compared with the second protocol in which starvation was performed with a medium containing 2% FBS through a stabilization process of 10% FBS-containing medium, ≪ / RTI >

e. After thawing process

Figure 4 shows the results for the first to fourth protocols for Experiment A, respectively. Specifically, Figure 4a, 4b and 4c respectively, cell density ^{4x10 4 cells / well, 5x10 4} cells / well, 6x10 4 cells / well ( each ^{2x10 5 cells / ml, 2.5x10 5} cells / ml, 3x10 5 cells / ml And then 0.2 ml / well of the solution was diluted to a final concentration per well).

Cell number and cell shape were found to be optimal until the BrdU assay proceeded with the fourth protocol of starvation with 2% FBS-containing medium immediately after thawing and without stabilization. When washed in the middle, it was confirmed that the cells were unstably separated and floated depending on the wells. This is one of the main causes of errors in the BrdU assay results.

In FIG. 4B, when the cell concentration was 5 × 10 ⁴ cells / well, cell number and cell shape were found to be the most suitable until the BrdU assay proceeded compared to other cell concentrations.

f. Sintering

Therefore, before proceeding with the BrdU assay, 4MBr-5 cells were seeded in 96-well plates at a concentration of 5x10 ⁴ cells / well, and then frozen in 80% F-12K medium containing 10% FBS and 10% DMSO In the case of defrosting, stabilization and starvation are carried out with F-12K medium containing 2% FBS immediately without washing, which is the most preferable embodiment.

3. BrdU Assay

The experiments performed so far are intended to confirm the complex conditions for reaching the log phase state after 4MBr-5 cells are well grown and proliferated in as short a time as possible after freezing and thawing.

The ultimate goal of the present invention is to perform a BrdU assay to determine whether the FGF7 recombinant protein has biological activity and the degree of biological activity. Thus, in this experiment, the frozen stock solution of a specific composition And the number of cells seeded per well were subjected to BrdU assays. Details of the experiment and results are as follows.

a. 4MBr-5 cells were cultured and frozen

4MBr-5 cells were inoculated into a 96-well plate at a concentration of 2.5x10 ⁵ cells / ml in 0.2ml / well of F-12K medium containing 10% FBS and 30ng / ml EGF and cultured for 1 day.

After confirming that the 4MBr-5 cells had stabilized to reach the exponential phase, the medium was exchanged with a frozen stock solution consisting of 10% DMSO, 10% FBS and 80% F-12K medium. Then, 4 MBr-5 cells were frozen in Deepfreezer at -70 ° C and stored for 1 week, 4 weeks, and 8 weeks, respectively.

b. Thawing of 4MBr-5 cells

To perform BrdU assays, 4MBr-5 cells, which were kept frozen for 1 week, 4 weeks, and 8 weeks, respectively, were thawed. When thawed as quickly as possible (preferably, 30 minutes within) 4MBr-5 cells were dissolved to avoid possible damage, washed just 2% FBS replaced with the F-12K medium containing 37 ℃ without process, 5% CO ₂ The starvation process was performed in the incubator. At this time, it is considered that the stabilization is performed simultaneously with the starvation process.

5 shows microphotographs of starvation 1 day after thawing and 4MBr-5 cells on day 2 after addition of FGF7 recombinant protein. As shown in FIG. 5, on the second day after FGF7 treatment, 4MBr-5 cells normally grow and proliferate to reach the exponential phase (log phase) irrespective of whether the freezing period is 1 week, 4 weeks or 8 weeks . In particular, it was observed that 4MBr-5 cells were well attached to the bottom of the plate. This was observed when compared with the control (FIG. 1) in which the starvation was performed with 2% FBS without freezing and the assay was performed , Shape, and aspect are similar.

Accordingly, it can be confirmed that the 4MBr-5 cells, which were frozen and stored by the method according to the present invention, can be used for the BrdU assay for measuring the activity of the FGF7 recombinant protein immediately after thawing.

c. Preparation of FGF7 Recombinant Protein Samples to Measure Differentiation Activity

The applicant transfected human FGF7 gene into CHO DG44 cells to prepare a CHO cell line that overexpresses human FGF7 recombinant protein. The rhFGF7 recombinant protein was expressed from the thus-produced CHO cell line and then obtained through a series of purification steps.

The BrdU assay was performed to determine whether the rhFGF7 recombinant protein thus obtained exhibited the proliferative activity of 4MBr-5 cells.

d. BrdU assay

In order to compare with the rhFGF7 recombinant protein produced by the Applicant in the above-mentioned step, commercially available FGF7 protein (speed biosystems) was purchased and used as a positive control.

As a first experiment, 4MBr-5 cells without freezing and thawing were used.

A BrdU assay was performed on the applicant's rhFGF7 recombinant protein and Speed Biosystems' FGF7 protein, respectively.

F-12K containing 2% FBS such that the concentration of FGF7 protein was 31.2 ng / ml, 15.6 ng / ml, 7.8 ng / ml, 3.9 ng / ml, 1.9 ng / ml, 0.9 ng / ml and 0.48 ng / Lt; / RTI > Then, the diluted FGF7 protein-containing medium was added to a 96-well plate containing 4 MBr-5 cells on the first day of culture in an amount of 0.2 ml per well. When FGF7 protein was treated with 4MBr-5 cells for 2 days, BrdU assay was performed using the BrdU cell proliferation assay kit (cell signaling) according to the manufacturer's protocol. The absorbance was then measured at a wavelength of 450 nM using an ELISA reader device from Perkinelmer.

The experimental results are shown in Fig. 6A. It can be confirmed that the rhFGF7 recombinant protein produced directly by the present applicant shows an activity tendency similar to that of the FGF7 protein commercially available from Speed Biosystems. In particular, the absorbance values measured for the rhFGF7 recombinant protein produced by the present applicant directly were within the range of ED ₅₀ = 1.5 to 7.5 ng / ml as described in the product description of the FGF7 protein of Speed Biosystems.

As a second experiment, the BrdU assay was performed on rhFGF7 recombinant protein produced directly by Applicant using 4MBr-5 cells thawed after 8 weeks of freezing. The BrdU assay proceeded in the same way as the first experiment.

As shown in FIG. 6B, the BrdU activity obtained after thawing 4MBr-5 cells which were stored for 8 weeks according to the present invention was very similar to the BrdU activity obtained using 4MBr-5 cells not frozen in the first experiment It can be confirmed that it is displayed in the form of a graph.

These results were obtained because the time taken to stabilize 4MBr-5 cells in freeze storage and to stabilize them for BrdU assay was very short within 3 days and there was little difference in growth between 4MBr-5 cells ≪ / RTI >

Therefore, when 4MBr-5 cells are stored frozen according to the present invention, they can be used in the BrdU assay as in the case of not being frozen, even if the frozen storage period is long.

e. Sintering

According to the experimental results, when 4MBr-5 cells were frozen by controlling the freezing stock solution composition, cell concentration, starvation process, and washing process of the specific composition according to the present invention, the BrdU assay 4MBr-5 cells can be thawed immediately before use.

Therefore, it is possible to confirm easily, accurately and promptly whether the newly produced FGF7 recombinant protein normally exhibits growth and proliferation activity of epithelial cells or epidermal cells.

In addition, even if the period of storage of 4MBr-5 cells by freezing is prolonged for a long period of time, there is a convenience that the results of the BrdU assay can be uniformly obtained.

Claims (8)

A method for performing the BrdU assay for measuring the 4MBr-5 cell proliferative activity of the FGF7 protein,
a) Freezing 4MBr-5 cells with a freezing stock solution consisting of 10% DMSO, 10% FBS and 80% F-12K medium, and storing until the BrdU assay is performed;
b) thawing the cells to be frozen in step a); And
and c) performing a BrdU assay by adding FGF7 protein to the cells thawed in step b). The method according to claim 1,
Further comprising the step of a-0) of culturing 4MBr-5 cells in F-12K medium supplemented with 10% FBS, prior to step a). The method according to claim 1,
Further comprising the step c-0) of culturing the cells in F-12K medium supplemented with 2% FBS without washing the thawed cells after step b). The method of claim 3,
Wherein the culturing of step (c-0) is performed for 1 day to 2 days. The method according to claim 1,
Wherein the period of storage of the frozen cells in step a) is from 1 day to 8 weeks. The method according to claim 1,
Wherein the cells are included in a multi-well plate in both steps a), b) and c). 3. The method of claim 2,
In step a-0), 4MBr-5 cells are cultured on a multi-well plate containing F-12K medium supplemented with 10% FBS at a concentration of 2 x 10 ⁵ to 3 x ¹⁰ cells / ml . delete

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