JP6218208B2 - Cell evaluation method and use thereof - Google Patents

Description

  The present invention relates to a cell evaluation method. More specifically, the present invention relates to a stem cell identification method and a cell evaluation method for transplantation using cell motility as an index. The method of the present invention is particularly suitable for quality evaluation of autologous cultured epidermis or autologous cultured corneal epithelium. The present invention is useful in fields such as regenerative medicine, for example.

  A cultured epidermal transplantation technique has been developed in which epidermal keratinocytes having high proliferative ability are obtained from normal skin, a sheet-like cultured epidermis is prepared by culture, and transplanted to a skin injury site of a patient. In the culture system of human epidermal keratinocytes used in regenerative medicine, it is important to evaluate the culture state and the content of cells having high proliferation ability. In addition, epidermis cultured for the purpose of transplantation requires quality control as a product unlike organ transplantation, which is a medical practice.

  Human epidermal keratinocytes form colonies under appropriate culture conditions (Non-patent Document 1), but keratinocytes that form colonies include several types of cells having different proliferative activities (Non-patent Document 2). ). Among these, human epidermal keratinized stem cells (also called holoclones: Non-patent document 2) have high proliferative activity and the ability to produce epidermal keratinocytes that humans need throughout their lives (non- Patent Documents 3 and 4). This culture of human epidermal keratinized stem cells has realized autotransplantation of a keratinocyte sheet to a wide range of severe burn patients (Non-patent Documents 5-7). This is widely recognized as a successful example of regenerative medicine using human stem cells (Non-patent Documents 8-11). Other keratinocytes capable of forming colonies are a progenitor cell having limited growth ability (Meroclone) and a cell having only transient growth ability (paraclone) (Non-patent Document 2). The former can regenerate the epidermis only for a short period of time, while the latter does not have that ability.

  Stem cell transplantation is essential for regenerative medicine of self-replicating tissues such as the epidermis. So far, human epidermal keratinized stem cells have been identified by clonal analysis (Non-Patent Document 2) and cell surface markers (Non-Patent Documents 12-14).

Rheinwald, J.G., & Green.H. Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells.Cell 6, 331-344 (1975). Barrandon, Y. & Green, H. Three clonal types of keratinocyte with different capacities for multiplication.Proc. Natl. Acad. Sci. USA 84, 2302-2306 (1987). Rochat, A., Kobayashi, K., & Barrandon, Y. Location of stem cells of human hair follicles by clonal analysis.Cell 76, 1063-1073 (1994). Mathor, M.B. et al. Clonal analysis of stably transduced human epidermal stem cells in culture.Proc. Natl. Acad. Sci. USA 93, 1031-1036 (1996). Gallico, GG, O'Connor, NE, Compton, CC, Kehinde, O., and Green, H. Permanent coverage of large burn wounds with autologous cultured human epithelium. N. Engl. J. Med. 311, 448-451 ( (1984). Pellegrini, G. Et al. The control of epidermal stem cells (holoclones) in the treatment of massive full-thickness burns with autologous keratinocytes cultured on fibrin.Transplantation 68, 868-879 (1999). Ronfard, V., Rives, JM, Neveux, Y., Carsin, H., & Barrandon, Y. Long-term regeneration of human epidermis on third degree burns transplanted with autologous cultured epithelium grown on a fibrin matrix.Transplantation70, 1588- 1598 (2000). De Luca, M., Pellegrini, G., & Green, H. Regeneration of squamous epithelia from stem cells of cultured grafts.Regen Med 1, 45-47 (2006). Green, H. The birth of therapy with cultured cells.Bioessays30, 897-903 (2008). Fuchs, E. The impact of cell culture on stem cell research.Cell Stem Cell 10, 640-641 (2012). Rochat A., Grasset, N., Gorostidi, F., Lathion, S., & Barrandon., Y. (2012) .Regeneration of epidermis from adult human keratinocyte stem cells.In Handbook of stem cells 2ed edition.Lanza R, Atala A, editors. Academic press, pp.767-780. Jones, P.H. & Watt, F.M.Separation of human epidermal stem cells from transit amplifying cells on the basis of differences in integrin function and expression.Cell 73, 713-724 (1993). Li, A., Simmons, P.J., & Kaur, P. Identification and isolation of candidate human keratinocyte stem cells based on cell surface phenotype.Proc. Natl. Acad. Sci. USA 95, 3902-3907 (1998). Jensen, K.B. & Watt, F.M.Single-cell expression profiling of human epidermal stem and transit-amplifying cells: Lrig1 is a regulator of stem cell quiescence.Proc. Natl. Acad. Sci. USA 103, 11958-11963 (2006).

  It is believed that the cells contained in the cultured tissue product for transplantation are not single. Since it is obtained from human tissue composed of a plurality of types of cells, various cells can be mixed. Moreover, even if the cells originate from the same type of cells, cells that have lost their intended function as a result of different differentiation during culturing may also be present. At present, there is no non-invasive method that can confirm the presence of stem cells during culture. Non-invasive identification of cultured human epidermal keratinized stem cells enables monitoring and quality control of epidermal keratinized stem cell culture, which improves engraftment of transplanted stem cells and long-term maintenance of regenerated epidermis after transplantation Leads to. Such techniques can also be applied to the selection of cells with high proliferative potential for therapy.

  Stem cells form a densely aggregated form, that is, a colony, in culture. This is a common phenomenon, but the dynamics of stem cells within the colony are not clear. The present inventors have now found that human epidermal keratinized stem cells cause collective movements and can analyze the movements to identify keratinized stem cell colonies. Specifically, the present inventors have found that the migration speed of cells in a colony has a positive correlation with the proliferation ability of cells contained in the colony. This indicates that cell motility is an important parameter for quality control in culture systems.

The present invention provides the following.
[1] A method for evaluating cells obtained from a subject in a culture system, the method comprising a step of evaluating the proliferation ability of a cell using a cell migration rate as an index.
[2] The subject has an injured tissue and a healthy tissue, and the cells obtained from the subject are used to produce an autologous cultured tissue obtained from the subject's healthy tissue and transplanted to the injured site. The method according to [1].
[3] The method according to [1] or [2], wherein the tissue is skin or cornea, and the autologous cultured tissue is autologous cultured epidermis or autologous cultured corneal epithelium.
[4] The method according to any one of [1] to [3], wherein the cell moving speed is an average moving speed of a plurality of cells in a specific region in the culture system.
[5] The method according to any one of [1] to [3], wherein the moving speed of the cell is replaced with a value based on a change in luminance at a specific pixel of the plurality of cell images that are interval-captured.
[6] The absolute value of the luminance difference in each pixel in the specific area is calculated from the two cell images taken with interval average imaging, and the total absolute value of the luminance difference in the specific area The method according to [4], wherein the motion index is divided by an area value of a specific area.
[7] A method for identifying a stem cell in a cell obtained from a subject, comprising a step of evaluating the proliferation ability of a cell by using the migration rate of the cell in a culture system as an index.
[8] The subject has an injured tissue and a healthy tissue, and cells obtained from the subject are used to produce an autologous cultured tissue to be transplanted to the injured site obtained from the subject's healthy tissue. The method according to [7].
[9] The method according to [7] or [8], wherein the tissue is skin or cornea, and the autologous cultured tissue is autologous cultured epidermis or autologous cultured corneal epithelium.

[10] A method for producing an autologous cultured tissue comprising:
Culturing cells obtained from a healthy tissue of a subject having a wounded tissue and a healthy tissue, and creating a self-cultured tissue for transplantation to the wounded site, and using the cell obtained from the subject in the culture system, using the migration speed as an index The manufacturing method including the process to evaluate.
[11] The method according to [10], wherein the autologous cultured tissue is autologous cultured epidermis or autologous cultured corneal epithelium.
[12] The method according to [10] or [11], wherein the moving speed is an average moving speed of a plurality of cells in a specific region in the culture system.
[13] The moving speed of the cell is calculated as a value based on a change in luminance at a specific pixel of a plurality of cell images obtained by interval imaging of cells in the culture system, according to any one of [10] to [12] the method of.
[14] The method according to [12], wherein the average moving speed of the plurality of cells is replaced with a motion index.

[15] An apparatus for carrying out the evaluation method according to any one of [1] to [6].
[16] An image input unit for inputting a plurality of cell images obtained by interval imaging of cells obtained from a healthy tissue of a subject having a wound tissue and a healthy tissue;
A motion index calculation unit that calculates a motion index from a plurality of input cell images;
A cell image analysis apparatus comprising:
[17] A threshold storage unit for storing a motion index threshold in advance;
The cell image analysis apparatus according to [16], further comprising a proliferative cell region specifying unit that compares the calculated motion index with a motion index threshold and specifies an area exceeding the threshold as a proliferative cell region.
[18]
An image input step for inputting a plurality of cell images obtained by interval imaging of cells obtained from a healthy tissue of a target having a wound tissue and a healthy tissue;
A motion index calculating step for calculating a motion index from a plurality of input cell images;
A program for running
[19] The computer includes a threshold storage unit that prestores a motion index threshold, compares the calculated motion index with the motion index threshold, and identifies a region exceeding the threshold as a proliferative cell region The program according to [18], wherein the program is further executed.

According to the present invention, it is possible to easily estimate the culture state and the content of highly proliferative cells in cells used for regenerative medicine such as human epidermal keratinocytes and cultured tissues prepared therefrom.
According to the present invention, culture conditions and stem cell proliferation can be evaluated non-invasively.

Correlation between migration and proliferation ability in human epidermal keratinocytes. a. Left panel: Motion analysis of growing colonies of human epidermal keratinocytes on 3T3 cells, which are feeder cells. Introduction of a simple and objective method using phase contrast microscope images for the management of cultured cells for regenerative medicine using images at 10-minute intervals (0, 10, 20 minutes). Converted to red, green and blue, all overlaid. The black and white part is the area where cells are not moving. Scale bar 50 mm. Right panel: The speed of movement of individual cells within the growing colony. Each point indicates the velocity of each cell in the colony. The vertical dots indicate the speed of cells in the same colony. The average speed every 30 minutes is indicated by a red dot. b. Left panel: terminal colony motion analysis. Right panel: The speed of movement of individual keratinocytes within the terminal colony. Scale bar 50 mm. c. Upper panel: Distribution of cell migration speeds inside and around the glowing colony. Bottom panel: Vector map of the growing colony. Each vector represents a trajectory of movement for 10 minutes. Scale bar 50 mm. d. Growing colonies show a variety of average migration speeds within the colonies. Scale bar 100 mm. e. Each growing colony of d was trypsinized and subcultured in a 10 cm culture dish. The top and bottom of d and e correspond. The culture dish was cultured for 12 days, fixed, and stained with rhodamine B. The growth activity of each growing colony was evaluated by the proportion of terminal colonies in a 10 cm culture dish after subculture. Scale bar 10 mm. f. Upper panel: Correlation between average cell migration rate and growth activity within each growing colony. Lower panel: Growing colonies divided into three groups based on average cell migration rate, and the difference in proliferative activity in each group. Each group is as follows. slow (≦ 30 mm / h), middle (30 mm / h <, ≦ 35 mm / h), and fast (> 35 mm / h). The p-value was calculated by Student's t-test. a. The combined experiment of time-lapse observation and clonal analysis was carried out 5 times independently. Experiments other than 20091107 showed a stronger correlation with respect to average cell migration rate and proliferation activity than the overall results. b. Measurements in all experiments. a. Each pixel of the phase contrast microscopic image of the cell has position information (X, Y) and luminance information (B). If the cell moves for a short time a, the luminance B of each pixel changes and ΔB is calculated. b. ΔB calculated from the phase contrast images of two colonies at 5-minute intervals and visualized with different brightness. The luminance at all the pixels in the colony was calculated (Σ | ΔB |), and averaged by the colony area (S) was taken as the motion index. c. The average cell migration rate in the growing colony and the terminal colony was 21.0 ± 9.76 mm / h (resulting from 86 cells in 7 colonies) and 6.55 ± 3.60 mm / h (resulting from 43 cells in 7 colonies, respectively). )Met. By calculating the motion index, we succeeded in expressing the difference in the collective cell movement speed. Effect of inhibitors on cell migration within a growing colony. 2-deoxy-D-glucose (2-DDG) was used at 50 mM. For the calculation method of the motion index, see the brief description of FIG. 3 and FIG. p values were calculated by Student's t test.

[Definition etc.]
In the present invention, when the range is represented by “n _{1 to} n ₂ ”, values at both ends are included unless otherwise specified.
The present invention provides a method for evaluating cells obtained from a subject in a culture system, the method including a step of evaluating the proliferation ability of the cells using the cell migration rate as an index. In the present invention, “culture system” refers to a state in which cells are maintained and cultured outside the body, unless otherwise specified.

  Regarding the origin of the cells cultured in the present invention, the term “subject” means animal organisms including humans, unless otherwise specified. Subjects include healthy subjects as well as subjects (patients) that are desirably treated by transplantation of cultured tissue. Subjects are those with skin defects due to burns, pressure ulcers (bed sores), ulcers, trauma, etc., or Stevens-Johnson syndrome, bullous keratopathy, keratoconjunctivitis, corneal ulcer, corneal herpes, corneal degeneration (dystrophy) ), A subject having damage to the cornea due to chemical wounds / burns or the like. If the subject has healthy tissue in addition to the damaged tissue, the cultured cells can be obtained from healthy tissue (eg, epidermis or limbus) derived from the subject itself including stem cells.

  In the present invention, the term “stem cell” refers to a cell having the ability to self-replicate and differentiate into a plurality of cell lineages unless otherwise specified. As used herein, the term “stem cells” includes skin stem cells, epidermal keratinized stem cells, retinal stem cells, retinal epithelial stem cells, cartilage stem cells, hair follicle stem cells, muscle stem cells, bone precursor cells, adipose precursor cells, hematopoietic stem cells, neural stem cells, liver Stem cells, pancreatic stem cells, ectoderm stem cells, mesodermal stem cells, endoderm stem cells, mesenchymal stem cells, ES cells, iPS cells, flat stratified epithelial cells (including tumor cells), induced epidermal angle Stem cells are included.

  INDUSTRIAL APPLICABILITY The present invention is particularly useful for application to epidermal keratinocytes (including epidermal keratinized stem cells) and cultured tissues (cultured skin sheets) prepared therefrom, and hereinafter, epidermal keratinocytes obtained from human skin. However, the present invention can be used for various cells and cultured tissues prepared from the cells. In addition to epidermal keratinocytes, the present invention is particularly useful for corneal epithelial cells and squamous epithelial cells (including tumor cells). In the following, the present invention may be explained by taking cells derived from the epidermis and cultured tissues obtained therefrom as examples, but the explanation also applies to cells derived from other tissues including stem cells, unless otherwise specified. apply.

[Evaluation / Identification Method]
The present invention relates to a method for evaluating cells obtained from a subject in a culture system, the method comprising a step of evaluating the cell growth ability using the cell migration rate as an index, and stem cells in cells obtained from the subject. Provided is a method for identification, which comprises a step of evaluating the proliferation ability of a cell using the moving speed of the cell in a culture system as an index. The present invention also provides a method for evaluating culture conditions suitable for maintaining proliferative properties, the method comprising a step of evaluating cell growth ability using the rate of cell migration in the culture system as an index. .

  The present inventors have found that, in a cultured human epidermal keratinocyte culture system, the average cell migration rate is maximum in colonies formed by highly proliferative cells, particularly colonies formed by keratinized stem cells, and humans It was found that the proliferation ability of epidermal keratinocytes correlates with the migration rate (Example and FIG. 1f). The present invention has been made based on such novel findings. In the present invention, when a cell has “proliferative ability” or “proliferative ability”, the cell can form a colony, and the formed colony loses the proliferative ability unless otherwise specified. This refers to the case where the cells are not composed of broken cells or cells whose growth ability is being lost, but are composed solely of cells capable of further proliferation.

  The presence or degree of proliferation ability of epidermal keratinocytes can be determined by various methods such as proliferation test by actually culturing, analysis of whether or not stem cells (clonal analysis, confirmation of the presence of cell surface markers), etc. Can be confirmed.

  Specifically, confirmation by culture can be performed as follows. Human epidermal keratinocytes form at least two types of colonies: a growing colony composed of small proliferating cells and a terminal colony composed of flat cells that are losing proliferative activity. (Non-Patent Document 11). Therefore, the frequency of appearance of terminal colonies composed of flat cells whose seeding keratinocytes are seeded at a cell density effective for cloning and started to grow, and which have lost their proliferative activity after culturing. Can be requested. The appearance frequency can be expressed as an appearance rate (%) (= number of terminal colonies formed / number of all colonies formed × 100).

  More specifically, the appearance rate (%) of the terminal colony can be obtained as follows. Inoculate the cells of interest in a culture dish at a cell density effective for cloning, and incubate under appropriate conditions for several days, for example, 12 days. For colonies formed as a result of the culture, stain if necessary. The terminal colony can be identified by morphological observation. One skilled in the art can morphologically distinguish terminal colonies from other colonies. Then, the terminal colonies are counted, and the ratio (%) of the terminal colonies to the total number of colonies can be obtained. In the study by the present inventors, in the growing colony, it was observed that the cells migrated spontaneously while maintaining a dense form (Example and FIG. The cells have been observed to be approximately quiescent (Examples and FIG. 1b).

  Analysis of whether or not it is a stem cell can be performed by clonal analysis or confirmation of the presence of a cell surface marker. For this method, see Non-Patent Document 2 and Non-Patent Documents 12-14. Can do. For confirmation of the presence of the stem cell marker, a commercially available anti-stem cell marker antibody can be conveniently used.

The cell migration rate (μm / h) can also be confirmed by various methods. For example, a cell in an appropriate environment can be appropriately measured by using a device for taking a time-lapse image attached to a conventional microscope and attached software. More specifically, the target cells are transferred to a chamber for microscopic observation, and conditions suitable for culturing the cells in the chamber, such as 37 ° C. and 10% CO ₂ , are set at intervals of several minutes, for example, intervals of 5 minutes. Then, take a time-lapse image over 180 minutes. The distance traveled for each 5 minutes is then tracked by comparing each image in turn. Then, the moving speed can be obtained by calculating the total moving distance in 180 minutes and dividing by the time.

  If necessary, the moving speed may be obtained for a plurality of cells (for example, 2 to 100 cells) in a certain area, and the average moving speed of the cells in that area may be obtained. In addition, the area | region here refers to the area | region which consists of the colony typically derived from one cell. Correlation between migration rate and growth ability was inoculated at a concentration effective for colony formation, and the average migration rate of cells in one colony formed away from other colonies and the colony were seeded with trypsin treatment Since it is sufficiently confirmed with the appearance rate (%) of terminal colonies in the system, the following is an example of evaluation of cells contained in one colony area formed apart from other colonies The present invention may be described in the following. However, even if colonies are formed in contact with or fused with other colonies, cell migration can occur within the colonies. Therefore, as an evaluation of cells contained in a region consisting of contact / fusion colonies identified by visual inspection or the like, or an area of about 100 to several hundred cells arbitrarily selected in confluently grown cells or sheet-like cells The present invention can be implemented. In the present specification, the migration speed may be explained with respect to cells contained in a region consisting of one colony formed away from other colonies, but the explanation is not limited to the case of special mention. This also applies to a region composed of colonies and cells included in an arbitrarily selected region.

  According to the study by the present inventors, when the experiment date is the same, the correlation between the moving speed and the proliferation ability may be clearer. This is probably due to the homogeneity of experimental conditions such as the passage number of 3T3 cells that are keratinocytes and feeder cells. Such matters may be noted when implementing the present invention.

  The average moving speed of cells contained in a certain area can be replaced by a motion index. The phase contrast microscope image of a cell has position information (X, Y) and luminance (B) information in each pixel (FIG. 3a). In the case of a phase difference image, a difference in intracellular structure appears as a difference in luminance. When cells move in a short time, the luminance at a certain pixel changes, so the present inventors have determined the absolute value of the luminance difference between the two colony phase difference images captured at an appropriate interval. (For real number a, a or its sign -a that is not negative is called the absolute value of a and is expressed as | a |), and the area of the colony (S) Average with. In the present invention, the term “motion index” refers to the value thus obtained (FIG. 3b). According to the present inventor, human epidermis keratinocyte colonies placed under low nutrient conditions by the addition of 2-DDG had a markedly reduced internal cell motility rate by actual measurement, The brightness difference of the continuous images (left figure) has been greatly reduced, and even with the indexing by the motion index, a significant difference was obtained (Fig. 3c).

  According to the motion index, an evaluation method combining a phase contrast microscope, a digital camera, and a simple image analysis program based on the present invention can be established.

  In the present invention, it is possible to perform cell identification, evaluation of culture conditions, evaluation of cultured tissue, and the like by cell movement speed (may be average movement speed) or motion index. It can be determined as appropriate. A preliminary test may be used to establish a baseline value (sometimes referred to as a “threshold”), and simultaneously control when determining cell migration rate or motion index for the target culture system. The same operation may be performed for the system, and the determination may be made by comparison with the value obtained for the control system.

  According to the study by the present inventors, under the conditions of the examples, for the growing colonies, the average moving speed is 21.0 ± 9.76 μm / h (n = 7), and the motion index is 28.5 ± 10.58 μm / h (n = 7). As for the terminal colonies, the average moving speed was 6.55 ± 3.60μm / h (n = 7) and the motion index was 11.3 ± 2.96μm / h (n = 7) (Figure 3). The determination criteria may be set with reference to these values. For example, when measured under the same conditions as in the examples, the average moving speed is 10.0 μm / h or higher or the motion index is 14.0 or higher, or the average moving speed is 12.0 μm / h or higher or the motion index is 17.0 or higher. May be used as a criterion.

  In our experiments using 2-deoxy-D-glucose (2-DDG), which inhibits processes related to cell motility, in the presence of 2-DDG, compared to the absence of 2-DDG, The motion index has been reduced to about 1/2 (Fig. 4), and this may be used as a reference for setting the judgment criteria. For example, when measured under the same conditions as in Examples, the kinetic speed or motion index is 1.2 times or more, or 1.5 times or more of the kinetic speed or motion index of the system using a sufficient amount of cell motility inhibitor. It is good also as a criterion.

  The motion index is calculated as a different value depending on the resolution of the image, exposure time, whether the image is 8-bit or 16-bit, etc., but the same image shooting is performed while maintaining the cells under the predetermined culture conditions. If it is performed under conditions, it can be expected that a judgment criterion that can be commonly applied in various cases can be obtained.

  Until now, there has been no method for non-invasively evaluating the culture conditions and the proliferation of cultured keratinocytes. However, according to the present invention that evaluates the cell growth ability using the cell moving speed as an index, noninvasive evaluation is achieved. In addition, the conventional management of culture conditions for maintaining the proliferation of stem cells was based on the experience of engineers. In the field of regenerative medicine, cells cultured for the purpose of transplantation require quality control as so-called products. Usually, the cells for preparing the transplanted tissue will vary depending on the individual from which it is derived, and the finished cell product may vary widely. Quality control of cultured tissue is extremely important. However, the calculation of the motion index of the present invention makes it possible to evaluate culture conditions and cells simply and objectively. In addition, quality control of cell products for regenerative medicine can be performed by introducing objective indicators that do not depend on the skill level of engineers.

[Method for producing cultured tissue]
In another aspect of the present invention, a method for producing a cultured tissue including an autologous cultured tissue is provided. In the present invention, “cultured tissue” refers to a tissue model that has been cultured in vitro using human cells and reconstructed unless otherwise specified. Examples of cultured tissues are cultured epidermis, cultured corneal epithelium, and cultured cartilage.

  The cultured tissue obtained by the production method of the present invention can be used for transplantation to a subject for symmetrical treatment. In addition, the cultured tissue obtained by the production method of the present invention can be used as an alternative to animals and simple cultured cells, and can be applied to various studies and experiments. For example, the cultured tissue can be applied to pharmacological tests, toxicity tests, and the like.

  The production method of the present invention includes at least a step (1): a step of culturing cells obtained from a subject to produce a cultured tissue, and a step (2): a cell obtained from the subject is cultured in a culture system, and the moving speed is an indicator At least the step of evaluating. Step (1) is typically a step of culturing skin keratinocytes obtained from a healthy skin of a subject having wounded tissue and healthy tissue, that is, a patient, and preparing autologous cultured skin for transplantation to the wounded site. In the following description, this case may be described as an example. However, those skilled in the art may appropriately modify the description to implement skin keratinocytes obtained from subjects other than patients, This can be applied to the case where it is performed on cells other than keratinocytes.

As step (1), when cultivating skin keratinocytes, a known method for culturing epidermal cells can be applied. A person skilled in the art can appropriately design the culture environment (including the medium, temperature, CO ₂ concentration, and culture period) according to the type of cell. If preferred, the cells of interest may be cultured on a layer of feeder cells such as 3T3 cells.

  The production method of the present invention includes, in addition to steps (1) and (2), a step of obtaining a tissue from a target, a step of separating and / or purifying a target cell from the tissue, and seeding the obtained cell in an appropriate culture environment Steps may be included. A step of removing the produced cultured tissue from the culture system may be included. The obtained sheet-like cells are transplanted into a patient. The process of obtaining tissue from the control can be a medical practice, but other processes can be performed by a person other than the physician.

  Step (2) of the present invention can be carried out without particular limitation before, during and after any of the above steps. From the viewpoint of determining whether or not the cells separated from the tissue are sufficient to produce a cultured tissue, the step (2) is preferably performed after the step of separating and / or purifying the cells. From the viewpoint of evaluating the quality of the cell tissue for transplantation, it is considered preferable to perform it before or before the step of removing the cultured tissue from the culture system during or after the step (1).

[Equipment, program]
The present invention also provides an apparatus and program for cell image analysis.

  Specifically, the apparatus includes an image input unit and a motion index calculation unit. Moreover, you may provide the threshold value memory | storage part and the proliferative cell area | region specific | specification part.

  The image input unit inputs digital data of a plurality of cell images obtained by interval imaging of cells obtained from a target healthy tissue having a wound tissue and a healthy tissue to a cell image analyzer. The input cell image may be an image of the entire cell sheet, an image of the entire culture system (for example, the entire culture dish) before sheeting, or an image of one colony. If it is an image of the entire cell sheet or an entire culture system, it may be an area where a motion index is specified (for example, an area corresponding to one colony), and the culture system before sheeting When inputting the entire image (for example, the entire culture dish), one or a plurality of regions that are calculated from the number of cells to several hundreds, and in which the motion index is to be calculated, may be designated. Hereinafter, the area where the motion index is calculated may be referred to as a “target area”.

  The motion index calculation unit calculates a motion index from a plurality of input cell images.

  The threshold storage unit stores in advance a motion index threshold used in the processing of the proliferative cell region specifying unit. Note that the motion index threshold can be determined as appropriate, as described above with respect to the determination criteria. A preliminary test may be performed prior to the analysis in accordance with the type and origin of the analysis target cell.

  The proliferative cell region specifying unit compares the calculated motion index with a motion index threshold, and specifies an area exceeding the threshold as a proliferative cell region in the whole or a part of the input cell image. Information indicating the position and range of the area may be acquired.

  The apparatus of the present invention may have an analysis result output unit. The analysis result output unit generates and outputs an analysis result based on the information about the specified region. The analysis result output unit may output the analysis result by displaying it on the image output device as a graphic such as a character or a graph, or may output it by printing it on a printer. The analysis results output by the analysis result output unit are, for example, the following items.

-Calculated motion index-Whether the colony or area is a proliferating cell area-Number of proliferating cell areas in the image-Area of each proliferating cell area and / or each proliferating cell area Ratio of total area / total area of cell area in image to total area of cell proliferation cell area

  An operation example of the cell image analysis apparatus of the present invention will be described. First, the image input unit inputs a plurality of digital images of cell images (here, two images will be described as an example) that have been interval-photographed into the cell image analyzer. Next, the motion index calculation unit calculates the absolute value of the luminance difference at each pixel in the target area in the two input images. Next, the motion index of the region is calculated by dividing the sum of the absolute values of the luminance differences calculated in the target region by the area of the target region.

  In some cases, the proliferative cell region specifying unit reads the motion index threshold stored in the threshold storage unit, specifies all regions having a motion index value greater than or equal to the threshold from the cell image, and ends the process.

  When the proliferative cell region specifying process ends, in some cases, the analysis result output unit generates and outputs an analysis result based on the specifying result, and ends the process.

  You may make it implement | achieve the function of the cell image analyzer mentioned above with a computer. In that case, a program for realizing each function may be recorded on a computer-readable recording medium, and the program recorded on the recording medium may be read into a computer system and executed. The “computer system” here includes an OS and hardware such as peripheral devices. The “computer-readable recording medium” refers to a portable medium such as a flexible disk, a magneto-optical disk, a ROM, a CD-ROM, or a storage device such as a hard disk built in the computer system. Furthermore, the “computer-readable recording medium” dynamically holds a program for a short time like a communication line when transmitting a program via a network such as the Internet or a communication line such as a telephone line. In this case, a volatile memory inside a computer system serving as a server or a client in that case may be included and a program that holds a program for a certain period of time. The program may be a program for realizing a part of the functions described above, and may be a program capable of realizing the functions described above in combination with a program already recorded in a computer system.

[Dynamics of epidermal keratinocytes in colonies]
Human epidermal keratinocytes form at least two types of colonies that are morphologically distinguishable. One is called a growing colony and is composed of small proliferating cells, and the other is called a terminal colony and is composed of flat cells that are losing their proliferative activity (see the above-mentioned non-patent document). 11). In the growing colony, cells were observed to migrate spontaneously while maintaining a dense form (FIG. 1a). In contrast, cells within the terminal colony were almost quiescent (FIG. 1b). The cell migration rate within the growing colony was 21.0 ± 9.76 mm / h (mean ± sd) (86 results for 7 colonies), and 6.55 ± 3.60 mm / h (mean ± sd) for the terminal colonies (7 The result was 43 cells in the colony (FIGS. 1a and 1b). In the growing colony, the moving speed of individual cells varied (FIG. 1a), and the cells inside the colony moved relatively faster than the edge of the colony (FIG. 1c).

  Human epidermal keratinocytes that form growing colonies exhibit different proliferative activities (Non-patent Document 2). Therefore, in order to confirm whether cell migration correlates with the proliferation ability of keratinocytes, the cell migration rate in the growing colony was measured by time-lapse observation, and then clonal analysis (Non-Patent Literature cited above). The proliferation ability was evaluated by 2) (FIGS. 1d and 1e). The proliferation ability of keratinocytes subcloned from individual colonies to a culture dish having a diameter of 10 cm was evaluated by the appearance frequency of terminal colonies appearing after culture. This analysis reveals that the frequency of terminal colonies that appear when subcloning colonies is negatively correlated with the rate of cell migration within the colonies before cloning (r = -0.477, p = 0.0335, n = 20), it was found that the colony composed of cells with high migration ability had higher proliferation activity (FIG. 1f). When the experimental date was the same, the correlation between the movement speed and the growth ability was stronger (Fig. 2). This is probably due to the homogeneity of experimental conditions such as the passage number of 3T3 cells that are keratinocytes and feeder cells.

  The above results indicate that keratinocyte colonies with extremely high proliferation activity derived from human epidermal keratinized stem cells (also referred to as holoclones) are composed of cells with high migration ability.

[Cell movement]
Experiments were conducted on the collective movement of keratinocytes in colonies by using low molecular weight compounds that inhibit processes related to cell movement. For this experiment, a method for evaluating collective cell migration within a colony was developed (a brief description of the drawings for FIGS. 3 and 3). By this method, a value (motion index) that was easily correlated well with the cell migration rate in the colony was obtained (FIG. 2). When intracellular ATP was depleted by 2-deoxy-D-glucose (2-DDG), keratinocyte migration within the colony was markedly inhibited (FIG. 4).

[Conclusion]
Maintenance and self-renewal of keratinized stem cells in a culture system are greatly involved in the success or failure of transplantation for epidermal regeneration (Non-patent Document 11). Here we show that human epidermal keratinized stem cell colonies are composed of cells with high migration ability. This discovery provides the basis for a non-invasive method to assess the ability of cultured keratinocytes to proliferate by analyzing cell motility and to assess the quality and monitoring of keratinocytes suitable for transplantation.

[Samples and experimental methods]
[Cell culture]
Epidermal keratinocytes (KURABO) isolated from human forensic foreskin were used. The keratinocytes were cultured under conditions of 37 ° C. and 10% carbon dioxide using 3T3-J2 cells (distributed by Professor Howard Green, Harvard University) treated with irradiation or mitomycin C as feeder cells. The composition of the culture is as follows: 10% fetal calf serum (FCS), 5 mg / ml insulin, 0.4 mg in a 3: 1 mixture of Dulbecco-Vogt modification of Eagle's medium (DMEM) and Ham's F12 medium / ml hydrocortisone, 10 ⁻¹⁰ M cholera toxin, and 2 × 10 ⁻⁹ M triiodothyronine (T3) were added (Non-Patent Documents 1 and 3 above). The medium was changed every 4 days, and the culture was finally fixed with 3.7% formalin and then stained with 1% rhodamine B. Colonies were observed and counted under a stereomicroscope. Clone analysis was performed according to the method described in Non-Patent Document 2 described above. 2-deoxy-D-glucose was purchased from Sigma-aldrich.

[Time-lapse observation and image analysis]
Keratinocytes were seeded at low density using 3T3 cells as feeder cells in a 35 mm culture dish and cultured for 6 or 7 days. Then, for time lapse observation, keratinocytes were transferred to a microscope observation chamber (ZEISS) set at 37 ° C and 10% carbon dioxide, and type lapse observation was performed with the Axiovert 200M microscope (Zeiss). . Images were acquired at 5-minute intervals for 180 minutes. From the time-lapse images, cell migration rates were calculated using MetaMorpho (Molecular Devices) or Volocity (PerkinElmer) on a graphic tablet (Wacom). The cell moving speed was calculated by tracking the moving distance for 5 minutes each and calculating the total moving distance in 180 minutes.

[Motion index calculation method]
There are 1300x1030 by acquiring phase difference images of two glowing colonies at 5 minute intervals and executing Adobe Photoshop image> Drawing mode of calculation command "Absolute value of difference" from two consecutive images The absolute value of the luminance difference between each pixel was calculated. Further, the motion index was obtained by averaging the absolute value of the difference in luminance at the pixels in the colony with the area value of the colony. The colony area was calculated using Volocity (PerkinElmer) on a graphic tablet (Wacom).

  In conventional cell culture relating to epidermal regenerative medicine, management of culture conditions for maintaining the proliferation of stem cells relies on the experience of engineers. Moreover, there has been no method for non-invasively evaluating the culture conditions and the proliferation of cultured keratinocytes. However, according to the present invention, a simple and objective method based on a phase contrast microscope image or the like can be introduced into cultured cell management.

  In the field of regenerative medicine, cells cultured for the purpose of transplantation require quality control as so-called products. Usually, the cells for preparing the transplanted tissue will vary depending on the individual from which it is derived, and the finished cell product may vary widely. Quality control of cultured tissue is extremely important. ADVANTAGE OF THE INVENTION According to this invention, quality control of the cell product for regenerative medicine can be performed by introducing the objective parameter | index independent of an engineer's skill level.

  According to the present invention, quality control of a cell product for regenerative medicine can be more easily performed by combining a phase contrast microscope, a digital camera, and a simple image analysis program based on the present invention.

Claims (12)

A method for evaluating cells obtained from a subject in a culture system, and evaluating the ability to form a colony composed of proliferating cells using the migration speed of multiple cells within the colony derived from the cells in the culture system as an index the step of viewing including,
The moving speed of multiple cells is the average moving speed of multiple cells,
Calculate the absolute value of the brightness difference at each pixel in the colony from the two colony images taken with interval imaging for the average moving speed of multiple cells, and calculate the total amount of brightness difference in the colony as the area value of the colony. A method that is replaced by the divided motion index . The subject has an injured tissue and a healthy tissue, and the cells obtained from the subject are for producing an autologous cultured tissue to be transplanted to the injured site obtained from the subject's healthy tissue. The method according to 1. 3. The method according to claim 2, wherein the tissue is skin or cornea, and the autologous cultured tissue is autologous cultured epidermis or autologous cultured corneal epithelium. A method for identifying epidermal keratinized stem cells or epithelial keratinized stem cells in cells obtained from the epidermis or epithelium of a subject, using as an index the migration rate of a plurality of cells in a colony derived from the cells in a culture system, the step of assessing the ability to form comprised colonies proliferating cells seen including,
The moving speed of multiple cells is the average moving speed of multiple cells,
Calculate the absolute value of the brightness difference at each pixel in the colony from the two colony images taken with interval imaging for the average moving speed of multiple cells, and calculate the total amount of brightness difference in the colony as the area value of the colony. A method that is replaced by the divided motion index . The subject has an injured tissue and a healthy tissue, and the cells obtained from the subject are for producing an autologous cultured tissue to be transplanted to the injured site obtained from the subject's healthy tissue. 4. The method according to 4 . 6. The method according to claim 5 , wherein the tissue is skin or cornea, and the autologous cultured tissue is autologous cultured epidermis or autologous cultured corneal epithelium. A method for producing an autologous cultured tissue comprising:
In a step of culturing cells obtained from a healthy tissue of a subject having a wounded tissue and a healthy tissue and producing an autologous cultured tissue for transplantation to the wounded site, and in a culture system, the colony derived from the cells obtained from the subject an indicator the moving speed of the plurality of cells, see contains the step of evaluating the fabricated autologous cultured tissue,
The moving speed of multiple cells is the average moving speed of multiple cells,
Calculate the absolute value of the brightness difference at each pixel in the colony from the two colony images taken with interval imaging for the average moving speed of multiple cells, and calculate the total amount of brightness difference in the colony as the area value of the colony. The manufacturing method that is replaced by the motion index divided . 8. The method according to claim 7 , wherein the autologous cultured tissue is autologous cultured epidermis or autologous cultured corneal epithelium. An image input unit for inputting a plurality of colony images obtained by interval imaging of colonies formed from cells obtained from healthy tissue of a target having a wound tissue and a healthy tissue;
Motion that calculates the absolute value of the brightness difference at each pixel in the colony from multiple input colony images, and calculates the motion index by dividing the total amount of brightness difference in the colony by the area value of the colony An index calculation unit;
A cell image analyzer for performing the evaluation method according to any one of claims 1 to 3 . A threshold storage unit for storing a motion index threshold in advance;
10. The cell image analysis device according to claim 9 , further comprising a proliferative cell region specifying unit that compares the calculated motion index with a motion index threshold and specifies an area exceeding the threshold as a proliferative cell region. Against the computer
An image input step for inputting a plurality of cell images obtained by interval imaging of cells in a colony derived from cells obtained from a healthy tissue of a target having a wound tissue and a healthy tissue;
Calculate the absolute value of the brightness difference at each pixel in the colony from the input cell images, and calculate the motion index by dividing the absolute value of the brightness difference in the colony by the area value of the colony.・ Index calculation step,
Ru is run, according to claim 1 to 3 any one program for carrying out the method for evaluating the. The computer includes a threshold storage unit for storing the motion index threshold in advance,
Comparing the calculated motion index and motion index threshold, Ru further execute a step of specifying a region exceeding the threshold value as proliferating cells region, the program of claim 11.