Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process...
Reexamination Certificate
2002-02-27
2004-11-09
Gitomer, Ralph (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
C435S029000
Reexamination Certificate
active
06815179
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method of measuring the stiffness of an in vitro cultured tissue for determining the transplant compatibility of the cultured tissue, a method of determining the transplant compatibility of the cultured tissue, a quality-control method for the cultured tissue, and a method of preparing a transplant-compatible cultured tissue.
2. Description of the Related Art
Regeneration medicine and tissue engineering, in which cells are seeded and cultured in vitro on a tissue regeneration scaffold (base material or support medium) and a tissue is regenerated to thereby reconstruct a human tissue, and the reconstructed cultured tissue is applied onto a living human body to thereby treat the living body have received attention in recent years. Such cultured tissue for transplantation must be examined for compatibility (suitability) for transplantation upon the application to the living body or must be examined for suitability for shipment upon shipment from a supplier.
As a possible candidate for methods for the determination of transplant compatibility, for the successive determination of the culture conditions or for the determination upon shipment (offering), a method of observing the structure of a cultured tissue is known, in which part of the cultured tissue is cut to thereby prepare tissue cross-sections, a produced matrix is stained with a stain, and the resulting chromatic figure is observed under a microscope.
However, it takes a long time to prepare tissue cross-sections for examination and such microscopic observation requires great effort from, and places a load on the examiner. Additionally, in order to obtain chromatic figures for each observation, this method requires a large quantity of test tissue pieces overall since part of the cultured tissue must be cut to prepare test tissue pieces, other cultured tissue (cultured tissue pieces of the same lot) must be cultured in the same conditions, etc.
Alternatively, an attempt has been made to verify the relationship between the amount of a produced matrix and the stiffness of the cultured tissue by a method of measuring the stiffness of the cultured tissue, in which the cultured tissue is processed with, for example, a cork borer and is subjected to a destruction test such as a compression test. Such a destruction test requires, however, a large quantity of tissue test specimens as in the observation of chromatic figures, since the destruction method requires the tissue test specimens of another cultured tissue of the same lot.
In addition, cell growth depends on the age or other conditions of a cell provider (cell donor) or on the nature of individual cells, and the degree of growth subtly varies from one cultured tissue to another, and therefore it is difficult to predict, for example, the time needed to become a culture tissue suitable for transplantation. For example, the time when the cultured tissue becomes compatible for transplantation cannot be significantly predicted. The difficulty in prediction of the culture inhibits precise culture control for bringing the cultured tissue into a desired condition. For these reasons, the quality of the cultured tissue upon provision cannot be significantly stabilized and a transplant-compatible cultured tissue cannot be reliably and easily prepared.
SUMMARY OF THE INVENTION
Under these circumstances, an object of the present invention is to provide methods of measuring the stiffness of a cultured tissue and of determining the transplant compatibility of the cultured tissue, in order to easily and nondestructively determine the transplant compatibility of the cultured tissue, to provide a quality-control method that can easily and appropriately control the quality of a cultured tissue, and to provide a method for reliably and easily preparing a transplant-compatible cultured tissue.
Specifically, the present invention provides, in one aspect, a method of measuring the stiffness of a cultured tissue for the determination of the transplant compatibility of the cultured tissue being cultured in vitro, which method is carried out by using a stiffness measuring device, the stiffness measuring device includes a detecting unit and calculation means, the detecting unit includes a contact unit, a vibrator connected to the contact unit, a vibration detecting unit for detecting vibration of the vibrator, where the calculation means determines stiffness information by calculation based on the detected result from the vibration detecting unit, and which method includes the steps of: bringing the contact unit into contact with the cultured tissue and measuring the stiffness of the cultured tissue.
According to the method of measuring the stiffness, the stiffness information of the cultured tissue is obtained from a change in frequency of the vibrator when the contact unit is contacted with the cultured tissue, and the stiffness of the cultured tissue is calculated from the stiffness information to thereby determine the amount of a matrix produced by the cultured tissue or to thereby determine the transplant compatibility based on the amount of the produced matrix.
Preferably, the detecting unit in the aforementioned method further includes a load detecting unit for detecting a load applied onto the contact unit, and the stiffness of the cultured tissue is measured based on a relationship between the vibration of the vibrator detected by the vibration detecting unit and the load detected by the load detecting unit.
According to this configuration, information of the load applied onto the contact unit is obtained, and the stiffness of the cultured tissue can be measured based on a relationship between the load information and the vibration of the vibrator to thereby further appropriately determine the transplant compatibility of the cultured tissue.
The detecting unit for use in the stiffness measuring method preferably further includes a displacement detecting unit for detecting a displacement of the contact unit from a reference position, and the stiffness of the cultured tissue is measured based on a relationship between the vibration of the vibrator detected by the vibration detecting unit and the displacement detected by the displacement detecting unit.
According to this embodiment, the contact unit is displaced from the reference position to yield a displacement, and the stiffness of the cultured tissue can be measured based on a relationship between the displacement and the vibration of the vibrator to thereby further properly determine the transplant compatibility of the cultured tissue based on its stiffness.
The cultured tissue for use in the measuring method preferably includes at least one of a cell and a matrix produced by the cell, which cell is seeded and cultured on a tissue regeneration scaffold having a three-dimensional structure, wherein the stiffness of the tissue regeneration scaffold alone on which no cell is seeded or the stiffness of the cultured tissue immediately after the seeding of the cell is previously measured and the resulting stiffness is defined as reference stiffness information, and the reference stiffness information is compared with the stiffness information of the cultured tissue.
According to this embodiment, the stiffness of the cultured tissue is measured by comparing the reference stiffness information with the stiffness information of the cultured tissue, which reference stiffness information is stiffness information of the tissue regeneration scaffold alone on which no cell is seeded. Alternatively, the stiffness information of the cultured tissue immediately after cell seeding can be used instead of the stiffness information of the tissue regeneration scaffold alone. This configuration can further appropriately determine the transplant compatibility of the cultured tissue based on its stiffness by comparing the same with the stiffness information of the tissue regeneration scaffold.
The tissue regeneration scaffold in the measuring method may include one selected fr
Fukushima Rika
Iwasa Junji
Katoh Masakazu
Kurushima Toyokazu
Ochi Mitsuo
Gitomer Ralph
Ochi Mitsuo
Sughrue & Mion, PLLC
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