JP2002296205A - Method of analyzing element composition of cultured tissue, method of determing implantation suitability and method of controlling quality, method of manufacturing cultured tissue having implantation suitability and cultured tissue having implantation suitability. - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a concrete index for determing the implantation suitability of a cultured tissue, and to provide the cultured tissue capable of being easily analyzed quantitatively and in distribution state of a product substrate of the cultured tissue, and having implantation suitability of stable quality. SOLUTION: The quantity and distribution of a specific element in the cultured tissue area analyzed with an electronic microprobe analyzer or the like to determine the implantation suitability of the cultured tissue. The implantation suitability of the cultured tissue is determined based on the quantity and distribution of the specific element in the cultured tissue. The quality of the cultured tissue is controlled while elementary analysis is conducted, and the cultured tissue having implantation suitability is manufactured.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an element composition analysis method for determining the suitability of a cultured tissue for transplantation, a method for determining a transplantability of a cultured tissue, a quality control method for a cultured tissue, and a cultured tissue having a transplantability. And a cultured tissue having transplantability.

[0002]

2. Description of the Related Art In recent years, attention has been focused on regenerative medicine and tissue engineering in which human tissues are reconstructed by cell culture in vitro (in vitro), and treatment is performed by applying the reconstructed cultured tissues to the living body again. I have.

[0003] When applying a cultured tissue reconstructed by cell culture to a living body, it is necessary to examine whether the cultured tissue is suitable as a tissue for transplantation. As one of such tests, there is generally known a staining method in which a substrate component produced from cells at the time of culture is stained with a staining agent, the staining state is observed, and the inspection is performed. For example, in the case of cartilage tissue, by staining acidic mucopolysaccharide, which is a substrate for producing chondrocytes, with Alcian blue, it is possible to determine whether or not the state is appropriate as a cultured tissue for transplantation. it can.

[0004] However, the inspection by the staining method does not provide quantitative measurement information, and the staining condition differs depending on the staining conditions such as the staining time, so that it is difficult to compare data. Needed. Further, when testing a plurality of types of production substrates, it is necessary to select a staining agent according to the target production substrate. Therefore, a large number of test tissue sections are required for each staining agent.

As another test method, a cultured tissue is dissolved with an enzyme or the like, and this is dissolved in a high-performance liquid chromatograph (HPL).
There is known a method for quantitatively measuring the composition of a cultured tissue by separating according to C).

However, according to this method, only the composition ratio of the whole culture can be obtained, and thus the distribution state and the like cannot be confirmed. In addition, the dissolving step for the quantitative measurement also requires time and effort. For example, in the case of cultured cartilage tissue, acidic mucopolysaccharides such as chondroitin sulfate and keratan sulfate can be cited as characteristic production substrates of chondrocytes.
Can only obtain quantitative analysis results of each production substrate (chondroitin sulfate, keratan sulfate, etc.). Therefore, in order to obtain the result as the whole production substrate (acid mucopolysaccharide), H
It takes time and effort to further process the results of quantification of each production substrate obtained by PLC.

[0007]

SUMMARY OF THE INVENTION In view of the above-mentioned problems of the prior art, the present invention provides a specific index for determining the suitability of a cultured tissue for transplantation and facilitates quantitative analysis of a substrate produced by the cultured tissue. The method for analyzing the composition of the cultured tissue and the method for determining the transplantation of the cultured tissue, which can also easily analyze the distribution state of the cultured tissue, the quality control method for the cultured tissue to provide a cultured tissue having stable quality transplantability, and It is an object of the present invention to provide a method for producing a cultured tissue having transplantability and a cultured tissue having transplantability.

[0008]

According to the first method of analyzing the elemental composition of a cultured tissue of the present invention, an electron beam irradiating means for irradiating the cultured tissue with an electron beam in order to determine the suitability of the cultured tissue for transplantation. An element analyzer comprising: a detection unit that detects X-rays emitted from the cultured tissue by the electron beam irradiation; and a measurement unit that measures an element composition of the cultured tissue based on the detection result. And measuring the elemental composition of the cultured tissue.

According to this method for analyzing the elemental composition of a cultured tissue, the elemental composition is analyzed based on the X-rays emitted from the cultured tissue by irradiation with an electron beam. Quantitative analysis and the distribution state of elements can be easily analyzed. Therefore, it is not necessary to prepare a staining agent for each substance to be measured or to use a plurality of cultured tissues themselves, and it is possible to objectively determine the suitability of transplanting cultured tissues objectively with a small number of materials.

In the second method for analyzing the elemental composition of a cultured cartilage tissue according to the present invention, the cultured cartilage tissue is irradiated with an electron beam in order to determine the suitability of the cultured cartilage tissue for transplantation. And detecting the sulfur (S) component contained in the cultured cartilage tissue based on the detection result.

According to this method, since the sulfur component is analyzed, for example, it is produced by differentiating cultured cartilage tissue or mesenchymal stem cells (MSCs) produced by reconstituting from chondrocytes. The objective quantitative analysis of the sulfur component in the cultured cartilage tissue and the distribution state in the cultured tissue can be easily analyzed, and the transplant suitability can be determined based on the analysis.

According to a third method for analyzing the elemental composition of a cultured tissue of the present invention, the cultured tissue is irradiated with an electron beam in order to determine the transplantability of the cultured bone tissue, and the cultured bone tissue is irradiated with the electron beam. The emitted X-rays are detected, and calcium (Ca) contained in the cultured bone tissue is determined based on the detection result.
Measuring the component or phosphorus (P) component.

According to this method, the calcium component or the phosphorus component is analyzed, so that the objective quantitative analysis of the calcium component and the phosphorus component in the cultured tissue and the distribution state in the cultured tissue are easily analyzed, and based on this, To determine transplant suitability.

[0014] The method for determining the suitability of a cultured tissue for transplantation of the present invention comprises:
It is characterized in that any one of the first to third elementary composition measuring methods for cultured tissues is used.

According to this method, any one of the first to third methods for measuring the elemental composition of a cultured tissue of the present invention is used for judging the suitability of the cultured tissue for transplantation. The transplant suitability of the cultured tissue to be measured can be easily and objectively determined based on the objective quantitative analysis and distribution of the components.

A first quality control method for a cultured tissue according to the present invention comprises an electron beam irradiating means for irradiating the cultured tissue with an electron beam, and a detecting means for detecting X-rays emitted from the cultured tissue by the electron beam irradiation. And an element analyzer comprising: a measuring unit for measuring an elemental composition of the cultured tissue based on the detection result.

According to this method, the quality of the cultured tissue is controlled based on the elemental composition based on the detection result of the X-rays emitted from the cultured tissue. The quality of the cultured tissue can be controlled appropriately and easily while judging the suitability for transplantation with a small number of materials based on the index.

According to a second quality control method of the present invention, the elemental composition of a cultured tissue is measured a predetermined time after the start of culture in order to determine the suitability for transplantation. The method is characterized by making predictions and adjusting a culture period or culture conditions of the cultured tissue based on the prediction results.

According to this method, the cultured tissue is cultured while adjusting the culturing period and the culturing conditions of the cultured tissue based on the time-dependent change of the predicted elemental composition. The quality of the cultured tissue can be appropriately controlled.

The method for producing a cultured tissue having transplant suitability according to the present invention is characterized in that the element composition of the cultured tissue is measured a predetermined time after the start of culture in order to determine the transplant suitability.

According to this method, the cultured tissue is produced while measuring the elemental composition in order to determine the suitability for transplantation.
Based on the specific criteria of elemental composition, it is possible to easily and surely produce a cultured tissue having transplantability.

Further, in the method for producing a cultured tissue according to the present invention, in the above-mentioned production method, the elemental composition of the cultured tissue is measured a predetermined time after the start of the culture, and the change with time of the specific elemental composition of the cultured tissue is predicted. And adjusting the culture period or culture conditions of the cultured tissue based on the prediction result.

According to the present invention, the cultured tissue is produced under the culturing period and the culturing conditions adjusted based on the elemental composition of the cultured tissue, so that the cultured tissue having transplantability can be easily and efficiently produced. it can.

Further, in the method for producing a cultured tissue of the present invention,
In the above manufacturing method, an electron beam irradiation means for irradiating the cultured tissue with an electron beam, a detection means for detecting X-rays emitted from the cultured tissue by the electron beam irradiation, and the cultured tissue based on the detection result And a measuring means for measuring the elemental composition of the present invention.

According to this method, the cultured tissue is produced while analyzing the elemental composition using the elemental analyzer.
A cultured tissue suitable for transplantation can be reliably produced while easily analyzing the elemental composition.

The cultured tissue having transplantability according to the present invention comprises an electron beam irradiating means for irradiating the cultured cartilage tissue reconstructed based on chondrocytes with an electron beam, and is released from the cultured tissue by the electron beam irradiation. When using an elemental analyzer comprising a detecting means for detecting X-rays and a measuring means for measuring the elemental composition of the cultured tissue based on the detection result, at least a part of the cultured tissue contains sulfur. (S) component is 0.1
０．４0.4% by mass.

According to the present invention, since a predetermined sulfur component is set as a criterion for judging the suitability for transplantation of a cultured tissue reconstructed based on chondrocytes, a culture containing a suitable amount of cartilage matrix and having high suitability for transplantation The organization can be provided reliably.

[0028]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Referring to FIG. 1, an element analyzer 10 according to the present invention will be described. The element analyzer 10 includes, above the stage 20 in FIG. 1, an electron gun 14 for emitting an electron beam L toward the stage 20, a converging lens 16 and an objective lens 1 disposed between the electron gun 14 and the stage 20.
And an electron beam irradiation unit 12 (corresponding to an electron beam irradiation unit of the present invention). Moving means (not shown) is connected to the converging lens 16 and the objective lens 18, and the focal position of the electron beam L can be adjusted by changing the position with respect to the stage 20.

An object M to be measured for elemental analysis can be placed on the stage 20. Also, on stage 20,
The stage 20 is moved in the X direction (for example,
A moving unit 22 composed of a drive motor or the like is connected so as to be movable in a direction (for example, a depth direction in FIG. 1) and a Z direction (for example, the vertical direction in FIG. 1).

The element analyzer 10 has an X-ray detector 24 for detecting characteristic X-rays generated from the object M to be measured.
(Corresponding to the detecting means of the present invention). The X-ray detector 24 is a wavelength dispersion type spectroscope (WDS: Wavelength D).
ispersive X-ray Spectrometer (EDS) and Energy Dispersive X-ray Spectr (EDS)
meter). In the case of a wavelength dispersion type spectrometer,
The X-ray detection unit 24 includes a spectral crystal, a spectroscope driving motor, and a detector. With this configuration, the X-ray detection unit 24 can detect only X-rays from electrons such as reflected electrons and secondary electrons and X-rays.

The X-ray detecting section 24 is connected to a control section 28 via an X-ray measuring system 26 (corresponding to the measuring means of the present invention). In the X-ray measurement system 26, characteristic X-rays derived from a specific element are extracted from the X-rays detected by the X-ray detection unit 24 by spectral analysis. In the control unit 28, the X-ray measurement system 26
Quantitative analysis and distribution analysis of elements in the measurement object M are performed based on the characteristic X-rays extracted in Step (1). Further, the control unit 28 is connected to the moving unit 22 connected to the stage 20, and can control the moving amount of the stage 20.

In the present invention, the cultured tissue cultured in vitro is placed on the stage 20 as the measurement object M of the element analyzer 10. Here, the culture tissue applicable as the measurement object M may be any tissue that has been reconstructed by in vitro culture and can be measured by the elemental analyzer 10, but may be identified as the culture period elapses. It is necessary that the tissue has an increased production of the substrate containing the element. Cells that reconstruct such cultured tissues include chondrocytes, osteoblasts, bone cells, fibroblasts, endothelial cells, epithelial cells, myoblasts, adipocytes, hepatocytes, nerve cells, or precursor cells thereof. And mesenchymal stem cells (MSCs). These cells may be cultured as they are, or precursor cells or MSCs may be cultured while being differentiated.

If a sufficient structure is maintained as a cultured tissue, a tissue regeneration carrier is not necessarily required. However, the cultured tissue to be measured is a tissue regeneration carrier having a three-dimensional structure. It is preferable to be composed of at least one of the cell and the production substrate held by the carrier for regeneration. This is because such a cultured tissue is suitable for transplantation into a living body in a form as it is. From the viewpoint of such transplantation form and ease of measurement, the cultured cells for which transplantation is determined according to the present invention include chondrocytes, osteoblasts, osteocytes or their precursor cells, mesenchymal stem cells (MSCs).
s) and the like are more preferable.

The tissue regeneration carriers applicable here include collagen, gelatin, hyaluronic acid, fibronectin, fibrin, chitin, chitosan, laminin, dermatan sulfate, heparan sulfate, chondroitin sulfate, calcium alginate, calcium phosphate, calcium carbonate, polycarbonate, and the like. Glycolic acid, polylactic acid, polyrotaxane and the like can be mentioned. Among these, collagen, gelatin, hyaluronic acid, and fibrin are more preferable from the viewpoint of compatibility with a living body and bioabsorbability.

When the cultured tissue is measured by the element analyzer 10, it is necessary to embed the cultured tissue. The embedding process is constituted by the steps of fixing, dehydrating, and embedding. Examples of the fixative used for fixation include glutaraldehyde, osmic acid and the like, and ethyl alcohol, acetone and the like can be used for dehydration. In each of these steps, a usual method can be applied as it is. Embedding can be performed under ordinary conditions using an epoxy resin or the like.

The measurement site in the measurement object M may be at least a part, and from the viewpoint of a culture for transplantation, it is preferable that the site that comes into contact with the living body at the time of transplantation be the measurement site.

The elements of the cultured tissue analyzed under such conditions vary depending on the type of the cultured tissue to be measured. In the case of the cultured cartilage tissue, the elements are largely contained in the cartilage matrix such as chondroitin sulfate. Sulfur (S) component, and in the case of cultured bone tissue, phosphorus (P) which is a component constituting the bone tissue
It is preferable to analyze the component, calcium (Ca) component. In the case of a cultured tissue that produces collagen, such as a cultured epidermal tissue or a cultured dermal tissue, it is preferable to analyze the nitrogen (N) component, which is one of the components constituting collagen. It is known that cultured cartilage tissue and cultured bone tissue also produce collagen, and analysis may be performed using nitrogen (N) components.

Some elements, such as nitrogen, can be constituents of the carrier. In such a case, the cell-derived element can be analyzed by the difference in the concentration, but it is preferable to use a carrier that does not contain the same elemental component or to prepare a cultured tissue without using the carrier. .

Next, the operation of the element analyzer 10 will be described. The culture tissue that has been embedded as a measurement target M is placed on the stage 20 and the moving unit 22 is controlled to
Adjust the position of and start the measurement. When you start measuring,
An electron beam L is emitted from the electron gun 14. The emitted electron beam L is converged by the converging lens 16, the focal position is corrected by the objective lens 18, and irradiates a part of the cultured tissue on the stage 20.

Electrons, X-rays and the like generated from the cultured tissue by irradiation of the electron beam L are detected by the X-ray detecting section 24, and the X-rays are counted. In the X-ray measurement system 26,
From the X-rays detected by the X-ray detection unit 24, characteristic X-rays derived from the element at the site irradiated with the electron beam are extracted, and the element composition corresponding to the characteristic X-rays is analyzed. With this elemental composition,
The distribution and quantity of elements at the irradiation site are analyzed. The element composition (concentration) is expressed as mass% at each measurement point,
The amount is converted from the X-ray count (X-ray intensity) based on a standard sample having a known elemental composition. It is also possible to use the X-ray count instead of the concentration by keeping the measurement conditions constant without converting the concentration (content) using the standard sample.

Further, by moving the stage 20 by the moving unit 22, the measurement is repeated while moving the analysis point on the object M to be measured in the analysis area, and the type and distribution state of the element in the analysis area can be determined. Measure. By expanding the measurement results obtained from such a plurality of analysis locations two-dimensionally, it is possible to obtain mapping data that enables the types and distribution states of the elements in the measurement region to be grasped.

The obtained mapping data can be subjected to image processing, whereby the types and amounts of elements can be displayed in different colors. In this case, it is possible to understand at a glance how the elements are distributed in the measurement region and the amount at each distribution location. For details of the element analyzer 10 as described above, see Japanese Patent Application Laid-Open No. 2000-2
No. 14111 (Title of Invention: Electronic Probe Microanalyzer) and the like.

In the present invention, by performing elemental analysis using the above-described elemental analyzer 10, the determination of the suitability for transplantation of the cultured tissue and the quality control are performed. <Judgment of transplant suitability> It is known that a tissue of a living body shows a specific elemental composition depending on each site, and produces a cellular factor / substrate having a specific elemental composition. A cultured tissue as a tissue equivalent used for transplantation is preferably in a state similar to such a living tissue from the viewpoint of physical properties and survival rate. For this reason, in the method for determining the suitability for transplantation of a cultured tissue of the present invention, the suitability for transplantation is determined based on the closeness to the living tissue by analyzing the elemental composition of the cultured tissue and the amount of substrate produced.

Hereinafter, the method for judging transplant suitability of the present invention will be described using a cultured tissue reconstructed based on chondrocytes as an example. FIG. 2 shows the results of elemental analysis of a rabbit knee joint cross section. As the element analyzer 10, an electron probe microanalyzer (hereinafter abbreviated as EPMA; manufactured by JEOL Ltd.) was used.

As shown in FIG. 2A, in the backscattered electron image, the bone part is displayed in white, whereas the cartilage part is almost a shadow part and cannot be discriminated. Therefore, even in the case of a knee joint section composed of a bone portion and a cartilage portion, the cartilage portion can be distinguished from a clear bone portion due to the unclearness of the cartilage portion. On the other hand, the image of the bone part is shown in a substantially uniform state.

On the other hand, as shown in FIG.
By analyzing and mapping with the S (sulfur) component, a cartilage portion rich in the S component and a bone portion hardly containing the S component can be clearly distinguished. In particular, when a color image is displayed, the S component can be more clearly identified by displaying the S component in blue, for example.

Although a slight S component can be observed in the bone part, its density is significantly different from the density of the S component in the cartilage part. The distribution can also be revealed. For example, in the color image of FIG. 2B, the cartilage portion is displayed in blue or yellow indicating that the concentration of the S component is high. Since the concentration is not as high as that of the part, the S component can be positioned as a characteristic element of the cartilage tissue. Note that, in FIG. 2B, a portion displayed in yellow is higher in density than blue.

In the case where a cultured tissue is prepared, it can be determined that if it is in a state equivalent to the state in a living body, it is sufficient for transplantability. In the case of cultured cartilage tissue, the S component in the measurement sample prepared based on the cartilage tissue of a living body is 0.1 to
Since it is generally distributed at a concentration in the range of 0.4% by mass, it is preferable that the concentration of the S component is at least 0.1% by mass and distributed around cells as a cultured tissue for transplantation. Also, too much S component (too much production substrate)
0.4% by mass
Less than is preferred.

As shown in FIGS. 2 (c) and 2 (d), the bone portion is analyzed by the P (phosphorus) component, and (d) is analyzed by the Ca (calcium) component. It can be clearly distinguished from the cartilage part. When displayed in a color image, the bone portion rich in the P component and the Ca component is displayed in red, whereas the cartilage portion hardly containing the P component and the Ca component is almost displayed as a shadow portion. Not done. As described above, since the cartilage portion and the bone portion can be clearly distinguished by the presence of the P component and the Ca component, the P component and the Ca component can be positioned as characteristic elements of the bone tissue.

Also, in the case of a color image, the density of the P component and the Ca component can be displayed by shading the color even in the bone portion. For example, when FIG. 2C is displayed as a color image, the display in pink is higher in density than in red. Thus, since the upper part of FIG. 2C is displayed in pink, it can be seen that the P component is more abundant than the lower part of FIG. 2C. On the other hand, when FIG. 2D is displayed as a color image, the colors are displayed in substantially the same color, and it can be seen that the Ca component is distributed at a substantially uniform concentration. Thus, the P component and Ca
Since the distribution and concentration of the components are displayed in an identifiable manner, they can be used alone or the transplantation suitability can be determined based on the correlation.

<Quality Control Method> In the present invention, quality control is performed based on the above-described composition analysis. In other words, in the cultured tissue, the substrate component produced from the cells tends to gradually increase with the culturing time. Therefore, the composition state of each cultured tissue is analyzed over time and monitored to grasp the culturing state. , Can be predicted.

By grasping and predicting the culture state in this way, it is possible to provide a cultured tissue in an appropriate state according to the state of the target transplant patient. For this reason,
Based on the specific index of composition analysis, it is possible to always provide a stable and high-quality cultured tissue for transplantation.
In addition, by extending or shortening the culture period or changing the culture conditions according to the culture state, cell growth can be promoted or delayed, and a predetermined cultured tissue can be provided at a desired time.

The measurement of the elemental composition for predicting the culture state depends on the growth rate of the cell type constituting the cultured tissue, but the more the number of times including the start of the culture increases, the more accurately the culture state can be predicted. If the culture condition can be grasped to some extent from the seeding density and the culture speed, the measurement may be performed at least once after the start of the culture. be able to. Thereby, quality can be managed more easily.

The adjustment of the culture of the cultured tissue is performed by lengthening or shortening the culture period or changing the culture conditions to promote or delay the cell growth depending on the state of the culture. When adjusting the cell growth rate, a change in the medium composition such as addition or removal of a nutrient factor, the time of medium exchange, the culture temperature, and the like can be mentioned. Thus, cell growth or substrate production of the cultured tissue can be promoted or delayed, and a cultured tissue in an appropriate state can be obtained at a desired time.

In the method for producing a cultured tissue having transplant suitability according to the present invention, a cultured tissue is produced while measuring the elemental composition used in the method for determining transplant suitability as described above. The measurement of the elemental composition is performed according to the production of the cultured tissue, and the cultured tissue having an appropriate elemental composition can be used as a cultured tissue having transplantability. For example, by measuring at the time of shipment during the culture period or immediately after the completion of the culture, it is possible to reliably provide only a cultured tissue having transplant suitability.

As described above, a cultured tissue can be produced while grasping and predicting the culture state based on the elemental composition. In this case, a time when an appropriate elemental composition is recognized is predicted from at least one measurement result after the start of the culture and the culture conditions. In addition, the culture period can be lengthened or shortened based on the prediction, or the culture conditions can be controlled, and the cultured tissue can be produced while adjusting the supply time. By continuing the culture until a predetermined time in this way, it is possible to reliably provide a cultured tissue having an appropriate elemental composition and suitable for transplantation with a small number of measurements.

[0057]

EXAMPLES Hereinafter, the present invention will be described with reference to an example of a cultured cartilage tissue reconstructed based on chondrocytes. Note that the element analyzer 10 was measured using the above-described EPMA.

<Preparation of cultured cartilage tissue> Articular cartilage was collected from the knee, hip and shoulder joints of Japanese white rabbits, and trypsin ED was used.
Enzyme treatment was performed with a TA solution and a collagenase solution, and chondrocytes were separated and collected. After washing the obtained chondrocytes,
10 v / v% FBS (fetal bovine serum) -DMEM (Dulbecco's modified Eagle's minimum essential medium) is added, and the cell suspension is adjusted to a cell density of 1 × 10 ⁷ cells / ml and 1 × 10 ⁸ cells / ml. Was prepared. The cell suspension and 3% atelocollagen implant (manufactured by Koken Co., Ltd.) are mixed at a ratio of 1: 4. 1 × 10 ⁷ cells / ml by this mixing process
And a cell density of 1 × 10 ⁸ cells / ml are diluted to 2 × 10 ⁶ cells / ml and 2 × 10 ⁷ cells / ml. 100 μl of this cell-collagen mixture was mounted (installed) on a culture dish.

The mounted mixture was heated at 37 ° C., 5% CO 2
_The mixture was allowed to stand for 0.5 to 1 hour under the conditions of ₂ to allow gelation, a medium was added, and the culture was started. The medium used was 10 v / v% FBS-DMEM containing 50 μg / ml ascorbic acid and 100 μg / ml hyaluronic acid.
Culture was performed for 3 weeks under the condition of% CO ₂ .

<Preparation of EPMA Sample> In order to examine the state of substrate production in the cultured cartilage tissue embedded and cultured in the collagen gel as described above, quantitative analysis of sulfur (S) component in the cross section of the cultured tissue using EPMA・ Mapping was performed.

Hereinafter, a method of preparing a measurement sample used in EPMA will be briefly described. After three weeks of culture, the cultured tissue was thoroughly rinsed (washed) with physiological saline, and then fixed with a 1% osmic acid solution for 1 hour. After fixation, wash with ion-exchanged water, immerse in 50% ethanol for 10 minutes to replace,
Dehydration was performed. 70%, 90%, 99.5%, 100%
And then gradually immersed in a highly concentrated ethanol to sequentially dehydrate. Furthermore, after substituting with n-butyl glycidyl ester (substituent for electron microscope), the culture tissue is measured by completely replacing with epoxy resin while gradually changing the ratio of n-butyl glycidyl ester to epoxy resin. As embedded. This sample was cut with a diamond disk, polished, carbon-deposited, and measured by EPMA.

<Measurement by EPMA> EPMA was measured at an acceleration voltage of 15 kV and an irradiation current of 0.05 μA. Fe: 46.55% by mass, S: 5 as a standard sample
3.45% by mass of FeS ₂ was used, and PE was used for the spectral crystal.
T (polyethylene terephthalate) was used. The measurement was performed with a probe diameter of 6.5 μm per point and a measurement time of 0.1 second, and 270 × 270 points (about 17 points).
An area of 55 μm × 1755 μm) was measured, and the result of the quantitative analysis of the S component was mapped.

<Comparison with EPMA and Staining Method> On the other hand, the distribution of acidic mucopolysaccharide, which is a chondrocyte production substrate, was examined by the staining method and compared with the EPMA composition analysis result (FIG. 3). The stained sample for comparison was formalin-fixed to cultured cartilage tissue cultured under the same conditions as the EPMA sample, frozen embedded, and then a tissue section was prepared and stained for acidic mucopolysaccharides (chondroitin sulfate, keratan sulfate, etc.). This was obtained by performing the Alcian Blue staining method in a conventional manner (FIG. 4). The presence of the acidic mucopolysaccharide can be confirmed by staining blue.

As shown in FIG. 4, from the Alcian blue stained image, the surface of the cultured cartilage tissue was 100 to 200 μm thick.
That a continuous layer of the production substrate is formed with a thickness of about, and cell colonies are formed inside the carrier,
It can be observed that a substrate is produced around it.
However, in Alcian blue staining, the base weight can be relatively estimated from the degree of staining, but the degree of staining is not clear, and it is difficult to grasp the actual base weight. For this reason, it is not preferable to use it as a standard for judging transplantability.

On the other hand, as shown in FIG.
From the result of the composition analysis of the S component by MA, 100 to 2
It can be observed that a large amount of the S component is distributed at a thickness of 00 μm, and that a portion having a large amount of the S component is distributed in a colony inside the carrier. The ratio of the S component in these portions was about 0.1 to 0.2% by mass. In the case of a color image, portions where the S component exists are displayed in blue or yellow, and in particular, portions where the S component is more abundant are displayed in yellow. From this color image, it can be seen that a higher concentration portion exists in the S component layer formed near the surface of the cultured tissue.

When this Alcian blue stained image is compared with the analysis image by EPMA, it can be seen that almost the same results can be obtained from both. From these results, it was shown that by analyzing the S component using EPMA, it is possible to obtain cartilage-specific matrix information as in the case of the Alcian blue staining method conventionally performed.

In addition, although the amount of the substrate cannot be quantitatively discussed in the Alcian blue staining method as described above, the quantitative analysis of the specific component is possible because the compositional analysis using EPMA is possible. Using this as a criterion, even a non-skilled person can easily examine the suitability of the cultured tissue for transplantation.

<Time-dependent change of production substrate by EPMA> Further, since a quantitative value can be measured by EPMA, the time-dependent change of the mass of the production base in culture can also be clarified. For this reason, it is possible to grasp the culture state of the cultured tissue based on such a change in the mass of the production base over time, control the culture by adjusting the length of the culture period, adjusting the culture conditions, and perform quality control of the cultured tissue. . 5 and 6 show the analysis results of mapping the S component distribution for each culture period. FIG. 5 shows the case where the seeding density at the start of culture was 2 × 10 ⁶ cells / ml, and FIG. Seeding density is 2 × 10 ⁷ / ml
belongs to. In both FIGS. 5 and 6, (a) is the first week of culture, (b) is the second week of culture, (c) is the third week of culture, (d) is the fourth week of culture, and (e) is the culture. The fifth week is shown.

FIG. 7 is a graph showing the change over time of the mass% of the S component. At this time, the EPMA measurement conditions were as follows: the probe diameter per point was 50 μm, and the measurement time was 20 μm.
0 seconds, n = 5 on the surface of the cultured tissue, and n =
Measured at 20 arbitrary points. The measurement inside the carrier was performed by taking 20 points in a 4 × 5 matrix at a position of 600 to 1000 μm from the surface of the cultured tissue. The other measurement conditions are the same as the measurement conditions at the time of mapping, and the data of FIG. 7 plots the average of each measurement point.

As shown in FIGS. 5 to 7, it was observed that the cells proliferated as the culture days passed, and the production substrate containing the S component specific to chondrocytes gradually increased with this. it can.

To explain this in detail, almost no S component was detected in the first week of culture, and it was found that the cartilage-specific matrix had not yet been produced. In the second week of culture, a large number of regions distributed in an oval shape within the collagen gel began to be observed, and it was found that a substrate with a concentration of S component of about 0.05% by mass began to be produced around the cells. In the third week of culture, more substrate is produced than in the second week, and a layer of a continuous production substrate is formed on the surface layer. At this time, the concentration of the S component was 0.1 to 0.2% by mass, which is sufficient for a cultured tissue for transplantation. In the fourth and fifth weeks of culture, it can be observed that the substrate is further produced on the surface of the cultured cartilage tissue, the concentration thereof increases, and the colony-like substrate distribution inside the carrier increases.

From the above, for example, when the average of each measurement point exceeds 0.1% by mass, it can be determined that the transplantation suitability exists. In addition, culture 5
Since the average of each measurement point exceeds 0.3% by mass even after the week, consider 0.4% by mass as the upper limit,
Transplant suitability can be easily determined.

In particular, in the color images shown in FIGS.
The state in which the concentration of the component is increasing is displayed in blue or yellow. In particular, as the number of culture days elapses, the number of blue indications indicating the presence of the S component inside the cultured tissue increases. At the same time, the yellow display indicating that the concentration of the S component is higher than that in the inside of the outer layer of the cultured tissue is increasing. It was also shown that such a tendency was more remarkable when the seeding density at the start of culture was higher.

Further, as described above, when the criteria for transplantability is 0.1 to 0.4% by mass, as shown in FIG. 7, when the seeding density is 2 × 10 ⁶ cells / ml, When the seeding density was 2 × 10 ⁷ cells / ml at the third week of culture, it was shown that at 2 weeks of culture, the cultured tissue was suitable for transplantation.

As described above, the culture condition can be grasped by performing the composition analysis of the cultured tissue with the lapse of time by EPMA, and the cultured tissue in an appropriate state can be provided according to the condition of the target transplant patient. And stable quality control can be performed. In addition, by changing or changing the culture period and culture conditions, cell growth can be promoted or delayed to obtain a predetermined cultured tissue at a desired time.

[0076]

As described above, according to the present invention, the quantitative analysis of the cultured tissue can be easily measured, and the transplant suitability of the cultured tissue can be easily examined. Further, by monitoring the composition analysis result of the cultured tissue over time, a cultured tissue having a stable quality can be provided.

[Brief description of the drawings]

FIG. 1 is a schematic diagram of an element composition analyzer according to an embodiment of the present invention.

FIG. 2 is a diagram showing the results of elemental analysis of a rabbit knee joint cross section by EPMA, (a) is a backscattered electron image, (b)
Is an image mapped by analyzing with the S component, (c) is an image mapped by analyzing with the P component, and (d) is an image mapped by analyzing with the Ca component.

FIG. 3 shows a cross section E of a rabbit knee joint according to an embodiment of the present invention.
It is the image which mapped the composition analysis result by PMA.

FIG. 4 is an Alcian blue stained image of a rabbit knee joint cross section according to an example of the present invention.

FIG. 5 is an image showing a time-dependent change in the distribution of S components in a cultured tissue which has been cultured at a seeding density of 2 × 10 ⁶ cells / ml.

FIG. 6 is an image showing a time-dependent change in the distribution of S components in a cultured tissue which has been cultured at a seeding density of 2 × 10 ⁷ cells / ml.

FIG. 7 is a graph showing the time-dependent change of the S component concentration in the cultured tissue after the start of the culture.

[Explanation of symbols]

 Reference Signs List 10 elemental analyzer 12 electron beam irradiation unit (electron beam irradiation unit) 20 stage 22 moving unit 24 X-ray detection unit (detection unit) 26 X-ray measurement system (measurement unit) 28 control unit L electron beam M object to be measured

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) // C12Q 3/00 C12R 1:91 4C097 (C12N 5/06 C12N 5/00 E C12R 1:91) ( 72) Inventor Takeyuki Yamamoto 6-209, Mitani Kitadori, Gamagori City, Aichi Prefecture Japan Tissue Engineering Co., Ltd. (72) Rika Fukushima 6-209 Mitani Kitadori, Gamagori City, Aichi Prefecture 1 Japan Tissue Co. Within engineering (72) Inventor Toyoichi Kururushima 6-209 Mitani Kitadori, Gamagori City, Aichi Prefecture Japan Tissue Engineering Co., Ltd. F-term (reference) 2G001 AA03 BA05 CA01 GA01 GA06 HA13 JA02 JA13 KA01 LA01 NA08 NA09 NA15 2G045 AA40 BB20 BB22 BB24 CB01 CB13 CB30 DB30 FA11 FA40 FB1 5 FB20 GC06 GC22 GC30 HA09 JA04 JA07 4B063 QA01 QQ02 QR72 QX01 4B065 AA93X BC50 CA44 4C081 AB04 AB05 CD34 DA12 4C097 AA03 BB01 DD15 MM04 MM05

Claims (10)

[Claims] 1. To determine the transplantability of a cultured tissue,
Electron beam irradiating means for irradiating the cultured tissue with an electron beam, detecting means for detecting X-rays emitted from the cultured tissue by the electron beam irradiation, and elemental composition of the cultured tissue based on the detection result A method for analyzing the elemental composition of a cultured tissue, wherein the elemental composition of the cultured tissue is measured using an elemental analyzer comprising: 2. In order to determine the suitability of the cultured cartilage tissue for transplantation, the cultured cartilage tissue is irradiated with an electron beam, X-rays emitted from the cultured cartilage tissue by the electron beam irradiation are detected, and the detection result is obtained. Measuring the sulfur (S) component contained in the cultured cartilage tissue based on the above-mentioned method. 3. In order to determine the suitability of the cultured bone tissue for transplantation, the cultured tissue is irradiated with an electron beam, and X-rays emitted from the cultured bone tissue by the electron beam irradiation are detected. Measuring the calcium (Ca) component or the phosphorus (P) component contained in the cultured bone tissue based on
A method for analyzing the elemental composition of a cultured tissue, comprising: 4. A method for determining the suitability of cultured tissue for transplantation using the method for measuring elemental composition according to claim 1. 5. An electron beam irradiator for irradiating a cultured tissue with an electron beam, a detector for detecting X-rays emitted from the cultured tissue by the electron beam irradiation, and a detector for the cultured tissue based on the detection result. A quality control method for a cultured tissue, comprising using an element analyzer having a measuring means for measuring an element composition. 6. The elemental composition of a cultured tissue is measured after a predetermined time from the start of cultivation in order to determine the suitability for transplantation, and a change over time of a specific elemental composition of the cultured tissue is predicted, based on the prediction result. A quality control method for a cultured tissue, comprising adjusting a culture period or a culture condition of the cultured tissue. 7. A method for producing a cultured tissue having transplant suitability, comprising measuring an elemental composition of the cultured tissue after a predetermined time from the start of culture in order to determine transplant suitability. 8. The elemental composition of the cultured tissue is measured at a predetermined time after the start of the culture, and the change with time of a specific elemental composition of the cultured tissue is predicted. Based on the prediction result, the culture period of the cultured tissue or The method for producing a cultured tissue according to claim 7, wherein the culture condition is adjusted. 9. An electron beam irradiator for irradiating the cultured tissue with an electron beam, a detector for detecting X-rays emitted from the cultured tissue by the electron beam irradiation, and the cultured tissue based on the detection result. The method for producing a cultured tissue according to claim 7, wherein an element analyzer comprising: a measuring means for measuring the elemental composition of the culture is used. 10. An electron beam irradiation means for irradiating a cultured cartilage tissue with an electron beam, a detection means for detecting X-rays emitted from the cultured tissue by the electron beam irradiation, and the cultured tissue based on the detection result. Measuring means for measuring the elemental composition of
When an elemental analyzer having the following is used, at least a part of the cultured tissue contains a sulfur (S) component of 0.1 to 0.4.
A cultured tissue having transplant suitability, characterized in that the cultured tissue is contained by mass%.