JP2003052395A - Transplant adequacy determination method - Google Patents

Abstract

(57) [Summary] [Problem] It is possible to easily determine whether transplantation is appropriate. SOLUTION: The transplantation suitability judging method of the present invention is a method wherein a cultured tissue after culturing is used as a measurement target in a tissue state without performing dispersion treatment, and an intracellular enzyme in the cultured tissue to be measured is reacted. Treats itself with an enzyme activity measurement reagent that is converted to a spectroscopically measurable substance, spectrophotometrically quantifies the converted substance, and determines the transplantation suitability of the cultured tissue based on the quantified result. Is what you do. In this transplantation suitability determination method, since the cultured tissue after culturing is used as a measurement target in a tissue state without performing dispersion treatment, the measurement target is not damaged by the dispersion treatment. In addition, the activity of the intracellular enzyme in the cultured tissue is quantified using an enzyme activity measurement reagent, but since this enzyme activity is correlated with the number of viable cells, the viable cell rate can be obtained as a numerical value as a result, There is no need to count the number of cells under a microscope.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for determining the adequacy of transplantation of cultured tissue.

[0002]

2. Description of the Related Art Conventionally, in order to observe the state of cultured tissue, a method of measuring the viability (Viability) and the colony forming ability (Colony Forming Ability) is known.

As a method for measuring the viable cell rate, a method is known in which an examiner measures the number of viable cells using a hemocytometer while observing the cultured tissue under a microscope. An example is a dye exclusion test using blue. In the dye exclusion test using trypan blue, the cultured tissue is first dispersed to prepare a cell suspension (dispersed cells),
Then, the cell suspension and trypan blue solution are mixed and then allowed to soak on a hemocytometer, and an examiner counts the number of blue-stained cells under a microscope. Since the cells that are blue-stained are dead cells, the viable cell rate is expressed as a percentage by dividing the difference between the total number of cells and the number of blue-stained cells (the number of viable cells) by the total number of cells.

On the other hand, in the method of measuring the colony forming ability, first, a cultured tissue is dispersed to prepare a cell suspension, and then the cells are serially diluted and each diluted solution is reseeded in a petri dish, and then placed in an incubator. After culturing, colonies are formed, then the cells are fixed, the colonies (cells) are stained with Rhodanil blue staining, and the examiner counts the number of colonies under a microscope.

[0005]

By the way, in determining whether or not the cultured tissue after culturing is suitable for transplantation, it is important that the cells in the cultured tissue are alive. It is conceivable that the transplantability is determined based on whether or not the measured value satisfies a predetermined reference value.

However, in the above-mentioned dye exclusion test using trypan blue, it is necessary to count the number of cells under a microscope, which is troublesome for an examiner and is troublesome in operation. In addition, since it is necessary to trypsinize the cultured tissue for a long time in order to obtain dispersed cells from the cultured tissue after culturing, the number of viable cells after being significantly damaged compared to the actual cultured tissue Therefore, it could not be said that the result was appropriate because it reflects the number of viable cells in the cultured tissue to be transplanted.

Further, in determining whether or not the cultured tissue after culturing is suitable for transplantation, it is necessary to maintain the proliferating ability of viable cells for survival after transplantation. It is also possible to measure the proliferative ability, that is, the colony forming ability, and judge the transplantability according to the degree of the colony forming ability.

However, the above-mentioned method for measuring the number of colonies by staining with Rhodanil blue has a problem that it is necessary to count the number of colonies under a microscope, which is troublesome for an inspector and is troublesome to operate.

The present invention has been made in view of the above problems, and it is an object of the present invention to provide a method for determining the adequacy of transplantation of a cultured tissue, which can easily determine the adequacy of transplantation.

[0010]

[Means for Solving the Problems, Embodiments of the Invention, and Effects of the Invention] The present inventors have studied various methods for determining the transplantability of a cultured tissue without measuring the number of cells or the number of colonies under a microscope. As a result, the present invention has been completed. That is, the first method for determining the adequacy of transplantation of a cultured tissue of the present invention is to measure a cultured tissue after culturing as a measurement target in a tissue state without performing a dispersion treatment, and measure intracellular enzymes in the culture target tissue. By reacting it is converted into a spectroscopically measurable substance by treatment with an enzyme activity measuring reagent, the substance after the conversion is quantified spectroscopically, and the cultured tissue of the cultured tissue is analyzed based on the quantification result. The suitability for transplantation is determined.

In this method of determining the transplantability, the activity of the intracellular enzyme in the cultured tissue is quantified spectroscopically by using an enzyme activity measuring reagent, and the enzyme activity is correlated with the number of living cells. Therefore, as a result, the number of viable cells can be identified with the number of living cells, and the transplantability is determined based on the number, so that the number of cells can be easily determined without the trouble of counting under a microscope. In addition, since the number of viable cells is checked as it is without damaging the cultured tissue, an appropriate result that reflects the number of viable cells in the cultured tissue to be transplanted, as compared to the case of examining the number of viable cells after giving damage. Is obtained. Therefore, according to this transplantability determining method, it is possible to easily and appropriately determine the transplantability.

The second method for determining the adequacy of transplantation of a cultured tissue according to the present invention is to re-seed and culture the dispersed cells obtained by subjecting the cultured tissue after the culturing to a dispersion treatment to form a colony, which is the object of measurement. By reacting the intracellular enzyme in the colony to be measured, it is converted into a spectroscopically measurable substance by itself, and then treated with an enzyme activity measuring reagent, and the substance after the conversion is spectroscopically quantified. The suitability for transplantation of the cultured tissue is determined based on the quantitative result.

[0013] In this method of determining the adequacy of transplantation, the activity of an intracellular enzyme in a colony is spectroscopically quantified and quantified using an enzyme activity measuring reagent, but this enzyme activity has a correlation with the number of colonies. Since it can be identified with the number of colonies, and the transplantability is determined based on the number, the number can be easily determined without counting the number of colonies under a microscope. Therefore, according to this transplantability determining method, it is possible to easily determine the transplantability.

In determining the transplantability of a cultured tissue, it is important that the cells are alive, and even if the cells are alive, the proliferative ability, that is, the colony forming ability is maintained if the engraftment after transplantation is taken into consideration. Since it is also important to perform the determination, it is preferable to carry out the determination by performing both the first and second methods for determining the adequacy of transplantation of cultured tissues of the present invention.

As the cultured tissue after culturing, a partially cultured cell sheet obtained by cutting a part of the cultured cell sheet may be used. The cultured cell sheet may be prepared by any method, but may be obtained, for example, by collecting a part of a patient's skin or oral mucous membrane and culturing it in a sheet form using a tissue engineering technique. Examples of the partially cultured cell sheet include punch pieces obtained by punching the cultured cell sheet.

Examples of the enzyme activity measuring reagent include a redox type reagent which is reduced by the reaction of intracellular dehydrogenase in the culture tissue to be measured and converted into a substance absorbing a specific wavelength, and A degradable fluorescence type reagent which is hydrolyzed by itself to be converted into a substance that emits fluorescence by reacting with an intracellular hydrolase in a target cultured tissue is preferable.

The redox type reagent is preferably a tetrazolium salt or AlamarBlue. As a tetrazolium salt, lactate dehydrogenase (LD
H) such as H) or the like that accepts hydrogen released by the action of intracellular dehydrogenase via an electron carrier and is reduced to formazane, and examples thereof include XTT, MTT, and WST.
One selected from the group consisting of -1 and WST-8 is preferred. The compound names for each abbreviation are shown in Table 1.

[0018]

[Table 1]

Here, the chain reaction of the nicotinamide adenine dinucleotide (NAD) -tetrazolium system is shown below.
Shown in formula In this formula 1, the electron carrier is PMS.
(5-methylphenadium methylsulfate), Meldola blue, 1-methoxy PMS and the like.
In this chain reaction, the substrate and NAD ⁺ react with each other by the action of dehydrogenase to produce an oxidant and NADH,
The electron carrier is reduced by this NADH to produce an electron carrier reduced product and NAD ⁺ , and the tetrazolium salt is reduced by this electron carrier reduced product to produce formazan and an electron carrier. Therefore, there is a correlation between the amount of formazan produced and the activity of dehydrogenase. On the other hand, since there is a correlation between the activity of dehydrogenase and the number of living cells, the number of living cells can be determined by quantifying formazan. Examples of such NAD-tetrazolium-based redox type reagents include Cell Counting Kit-8 (including WST-8 and 1-methoxy PMS) and Cell Counting Kit (manufactured by Dojindo Laboratories). WST-1 and 1-methoxy PMS are included) and the like.

[0020]

[Chemical 1]

As the degradable fluorescence type reagent, an ester group-containing fluorescein which becomes an fluorescein which is hydrolyzed by intracellular esterase to fluorescein is preferable. Examples of such ester group-containing fluorescein include, for example, calcein-AM (Calcein A).
M) is preferred. This compound name is also shown in Table 1 above. Examples of such a degradative fluorescence type reagent include Cell Counting Kit-F (calcein-AM / DMSO solution) manufactured by Dojindo Laboratories.

[0022]

EXAMPLES Comparative Example 1. Evaluation of transplantation suitability based on dye exclusion test with trypan blue (1) Preparation of cultured epithelial cell sheet About 1 cm from a patient who gave informed consent
^Two skin tissues were collected. The collected skin tissue was treated with an epithelial detachment medium to separate it into a dermis layer and an epithelial layer, and this epithelial layer was trypsinized to prepare an epithelial cell suspension. Next, feeder cells were laid on the bottom surface of the culture flask, and then an epithelial cell suspension was seeded. Mouse fibroblasts whose growth ability was suppressed by mitomycin C were used as feeder cells. 5 after cell seeding
V / v% FBS (fetal bovine serum) -DMEM (Dulbecco's modified Eagle medium) was injected as a culture medium, and epithelial cells were cultured in the presence of feeder cells. After confirming the colony formation of epithelial cells, by continuously culturing, it becomes a stratified (sheeted) through a confluent state,
A cultured epithelial cell sheet was formed. Further, by the time the cultured epithelial cell sheet was formed, most of the feeder cells were excluded from the culture system.

(2) Measurement of viable cell rate As a measurement sample for enzyme activity measurement, the cultured cell sheet prepared as described above was 6 ° C, 12 ° C, 25 ° C, 3 ° C.
Three samples were stored at a constant temperature of 0 ° C., and the second, fourth, eighth, fifteenth, and thirtieth days after storage were used as measurement samples. On the 15th and 30th days, only 2 cases were used as measurement samples. The measurement was performed by the following procedure. That is, the cultured cell sheet was washed with PBS (phosphate buffer solution), then shredded, 0.1% trypsin was added thereto, pipetting was performed, and the mixture was allowed to stand at 37 ° C for 7 minutes, and then pipetting again. After that, the mixture was allowed to stand at 37 ° C. for 7 minutes, after which a serum medium was added to stop the reaction, and pipetting was performed again to give a cell suspension. A trypan blue solution prepared in advance was added to and mixed with the cell suspension, and the mixed solution was immediately soaked in a hemocytometer, and the number of blue-stained cells was counted under a microscope. The viable cell rate was calculated by the equation (1). In addition, a graph showing the change in the viable cell rate with respect to the number of storage days is shown in FIG. The viable cell rate in FIG. 1 was the average value of 3 cases. However, the viable cell rate on the 15th and 30th days was the average value of the two cases.

[0024]

[Equation 1]

(3) Judgment of transplantation suitability The number of cases suitable for transplantation judgment based on the viable cell rate obtained in the dye exclusion test with trypan blue is shown in Table 2.
It was shown to. Here, the transplantation appropriate standard value (“the compatible value” in FIG. 1) was set to 25% of the viable cell rate, and those exceeding this standard value were made the compatible examples. In Table 2, the number of measurement examples was used as the denominator, and the number of compatible cases was used as the numerator.

[0026]

[Table 2]

Example 1. Evaluation of transplantation suitability based on enzyme activity measurement (1) Preparation of cultured epithelial cell sheet 1. A cultured epithelial cell sheet was prepared in the same manner as in.

(2) Enzyme activity measurement As a measurement sample for enzyme activity measurement, the cultured cell sheet prepared as described above was 6 ° C, 12 ° C, 25 ° C, 3 ° C.
The samples were stored at a constant temperature of 0 ° C., and the samples on the 2nd, 4th, 8th, 15th, and 30th days after storage were used as measurement samples. The measurement was performed by the following procedure. That is, a cultured cell sheet to be used as a sample was punched at 12 locations with a size of 8 mm, and 4 punch pieces were stored in each well in a 48-well plate. At this time, 0.5 ml of the epidermal culture medium was injected into each well in advance. In this way, three measurement samples (three wells) under the same conditions were set. This 48-well plate was placed in a CO ₂ incubator for 3
After culturing for 2 hours under the conditions of 7 ° C. and 10% CO ₂ , 0.3 ml of the medium is sucked from each well and the remaining amount of the medium is 0.2
ml. Then WST-1 and 1-methoxy PMS
2 solution of the above cell counting kit containing
0 μl was added and shaken, and the cells were cultured in a CO ₂ incubator at 37 ° C. and 10% CO ₂ for 2 hours. During this culture, the cells were shaken again after 1 hour. Then, after culturing for 2 hours, the 48-well plate was cooled, and then the supernatant of each well was 110 μm with a micropipette.
1 was sampled and injected into a 96-well plate. Then, the absorbance of WST-1 formazan (measurement wavelength 450 nm, reference wavelength 650 nm) in each well of the 96-well plate was measured using a plate reader, and the absorbance (ΔOD obtained by subtracting the blank value of the medium alone was measured.
_{450-650 nm} / hr, absorbance per hour of reaction,
OD / hr). The result is shown in FIG. The measurement result was the average value of the absorbance of 3 wells. However, the measurement results on the 15th and 30th days except the storage temperature of 30 ° C are the average value of the absorbance of 2 wells, and the storage temperature is 3
The measurement was stopped at 0 ° C until the 15th day, and the measurement result on the 15th day was the absorbance of one well.

In FIG. 2, a sharp decrease in LDH enzyme activity can be observed immediately after storage, but after 15 days of storage, LD
It can be seen that the H enzyme activity is stable. In particular, it can be seen that the LDH enzyme activity is maintained at the highest when the storage temperature is 12 ° C. Further, it can be seen from the shape of the graph that the changes over time at all storage temperatures show the same tendency.

(3) Judgment of transplantation suitability Table 3 shows the number of compatible cases when the transplantation adequacy was judged based on the above enzyme activity measurement. Here, 0.012 (ml / cm ² ) * (O
D / hr) (Note: the absorbance when 1 cm of the sheet was reacted in 1 ml for 1 hour) was judged to be conforming, the result was in good agreement with the number of conforming examples in Comparative Example 1. Was used as the transplantation judgment standard value (“adaptation value” in FIG. 2). In Table 3, the number of measurement examples was used as the denominator, and the number of compatible cases was used as the numerator.

[0031]

[Table 3]

In this example, the LDH enzyme activity in the punch pieces of the cultured cell sheet was quantified spectroscopically and quantified using a cell counting kit, which is an enzyme activity measuring reagent. Can be identified with the number of living cells as a result because it correlates with the number of living cells, and the transplantability is determined based on that number, so the number of cells can be easily determined without the trouble of counting under a microscope. In addition, in the present example, by appropriately setting the transplantation determination standard value (transplantation determination standard absorbance), the same result as in the case of determining the transplantability based on the result of the conventional dye exclusion test with trypan blue was obtained. To be Furthermore, since the number of viable cells is measured by directly using the punched piece of the cultured cell sheet as a measurement target, it is more appropriate to reflect the number of viable cells in the cultured tissue to be transplanted than when the number of viable cells is examined after damage is given. Results are obtained.

Comparative Example 2. For microscopy (also called microscopy)
Confirm colony forming ability Trypsin treatment during dye exclusion test with trypan blue
Treated cell suspension to the same density as the cultured epithelial sheet.
48 well plate (0.75 cm ²/ We
Re-seeding and culturing for 1 week to form colonies
It was Then stain the colonies with Rhodanil blue
Then, the number of colonies was counted while observing under a microscope. Na
Oh, I measured 3 cases (No. 1-3) for each storage temperature.
It was The results are shown in Table 4. As is clear from Table 4
In addition, on the second day after storage, all cases maintained colony forming ability.
I was there. Also, at a storage temperature of 6 ° C, store until the 4th day after storage.
At a temperature of 12 ℃, 30 days after storage, at a storage temperature of 25 ℃
Up to 15 days after storage, 2 days after storage at 30 ° C
Until now, all cases showed colony formation. Also, the toe in Table 4
In the tal column, the number of measurement cases is used as the denominator
Is a colony-forming agent).

[0034]

[Table 4]

Example 2. Colony shape by enzyme activity measurement
Confirmation of ability After forming colonies in the same manner as in Comparative Example 2,
Confirmation of colony forming ability with cell counting kit
An approval test was conducted. That is, the well was replaced with 200 μl of fresh medium
And preincubated for 30 minutes
Then add 20 μl of the cell counting kit solution.
Added Incubate for exactly 1 hour, then cool, then culture medium
Absorbance (measurement wavelength 450
nm, reference wavelength 650 nm), and culture medium alone
Absorbance (△ OD _450-650nm/ Hr,
However, the average value of 3 wells (also referred to as OD / hr) is calculated.
It was The result is shown in FIG.

Table 5 shows the number of compatible cases when the transplant appropriateness was determined based on the above enzyme activity measurement. Here, for the absorbance of 3 wells (No. 1-3) at each storage temperature,
When the number of samples that exceeded 0.05 (ml / cm ² ) * (OD / hr) was determined to be compatible, the number of compatible examples in Comparative Example 2 was very well matched. “Cut line” in 3). In the total column of Table 5, the number of measurement examples was used as the denominator, and the number of compatible cases was used as the numerator.

[0037]

[Table 5]

In this example, LD in a colony was prepared using a cell counting kit which is an enzyme activity measuring reagent.
H enzyme activity was quantified spectroscopically and quantified.
Since the DH enzyme activity has a correlation with the number of colonies, as a result, it can be identified with the number of colonies, and the appropriateness of transplantation is determined based on the number, so that the number of colonies can be easily determined without the trouble of counting under a microscope. . In addition, in this example, by appropriately setting the transplantation determination reference value (transplantation determination reference absorbance), a transplantation appropriateness determination result equivalent to that obtained when counting the number of conventional colonies can be obtained.

It should be noted that the present invention is not limited to the above-described embodiments, and it is needless to say that the present invention can be implemented in various forms without departing from the technical scope of the present invention.

[Brief description of drawings]

FIG. 1 shows a dye exclusion test using trypan blue.
It is a graph showing the change of the number of preservation days and the viable cell rate.

FIG. 2 is a graph showing changes in the number of storage days and the absorbance (viable cell rate) in enzyme activity measurement.

FIG. 3 is a graph showing changes in storage days and absorbance (colony forming ability) in enzyme activity measurement.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Ikazu Masuwa             1-209-6-chome, Mitanikitadori, Gamagori-shi, Aichi               Japan Tissue Engineering Co., Ltd.             In nearing (72) Inventor Riki Shinohara             1-209-6-chome, Mitanikitadori, Gamagori-shi, Aichi               Japan Tissue Engineering Co., Ltd.             In nearing F-term (reference) 2G045 AA24 BA14 BB20 BB25 CB01                       DA20 FA16 FB01 FB11 FB12                       GC10 GC22                 4B063 QA05 QQ08 QQ24 QQ61 QR04                       QR41 QR64 QR77 QX01

Claims (10)

[Claims] 1. It is possible to measure spectroscopically by subjecting a cultured tissue after culturing to a measurement target in the tissue state without performing a dispersion treatment and reacting an intracellular enzyme in the culture target tissue to be measured. A method for determining the adequacy of transplantation, which is treated with an enzyme activity measuring reagent that is converted into a substance, spectroscopically quantifies the substance after the conversion, and determines the transplantability of the cultured tissue based on the quantification result. 2. The dispersed cells obtained by subjecting the cultured tissue after the culturing to the dispersion treatment are re-seeded and cultured to form colonies to be a measurement target, and the intracellular enzyme in the colony as the measurement target is reacted. By itself, it is treated with an enzyme activity measuring reagent that is converted into a spectroscopically measurable substance, the substance after the conversion is spectroscopically quantified, and the transplantability of the cultured tissue is determined based on the quantitative result. A method for determining the appropriateness of transplantation. 3. A transplant adequacy determination method using the transplant adequacy determination method according to claim 1 and the transplant adequacy determination method according to claim 2. 4. The transplantation adequacy determining method according to claim 1, wherein the cultured tissue after the culture is a partially cultured cell sheet obtained by cutting a part of the cultured cell sheet. 5. The redox-type reagent, wherein the enzyme activity measuring reagent is reduced by itself by reacting with intracellular dehydrogenase in the cultured tissue to be measured and converted into a substance absorbing a specific wavelength. 5. The transplant adequacy determining method according to claim 1, wherein 6. The transplantability determining method according to claim 5, wherein the redox reagent is a tetrazolium salt. 7. The tetrazolium salt is XTT or MT.
The transplant adequacy determination method according to claim 6, which is one selected from the group consisting of T, WST-1 and WST-8. 8. The degradable fluorescence type reagent, wherein the enzyme activity measuring reagent is converted into a substance which is hydrolyzed by itself by reacting with an intracellular hydrolase in the cultured tissue as the measurement target. 5. The transplant adequacy determining method according to claim 1, wherein 9. The transplantation adequacy determining method according to claim 8, wherein the degradable fluorescent reagent is an ester group-containing fluorescein. 10. The transplantation adequacy determining method according to claim 9, wherein the ester group-containing fluorescein is calcein-AM.