Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Primate cell – per se
Reexamination Certificate
1999-10-15
2003-01-14
Saucier, Sandra E. (Department: 1651)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Primate cell, per se
C435S366000, C435S370000, C435S377000, C435S383000, C435S353000
Reexamination Certificate
active
06506599
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method for culturing the Langerhans islets suitable for transplantation. More particularly, the present invention relates to a culturing method by which the Langerhans islets can be proliferated in volume, and the fact that proliferated islet autotransplantation can stimulate islet regeneration via islet replication and neogenesis, leads to a perfect diabetes cure.
2. Description of the Prior Art
Diabetes mellitus (usually referred to simply as diabetes) is a complex disease characterized by a grossly abnormal pattern of carbohydrate metabolism resulting from impaired insulin secretion and/or effectiveness. The incidence of diabetes in industrialized countries is about 10%. Indeed, diabetes is the most common serious metabolic disease in the world, it affects hundreds of millions.
Diabetes may be classified as insulin-dependent diabetes or noninsulin-dependent diabetes. An absence of or insufficient intrinsic insulin is a characteristic of insulin-dependent diabetes. Some diabetics have a normal or even higher than normal level of insulin in their blood, but they are quite unresponsive to the hormone. This form of the disease, known as non-insulin-dependent diabetes, typically develops later in life than does the insulin-dependent form. However, the diabetes-causing mechanism with which these two types can be discriminated has yet to be revealed.
For treatment, insulin-dependent diabetics should continue to receive exogenous insulin because their capacity of producing insulin is greatly lowered. However, it is virtually impossible to continuously and properly provide insulin in response to patient's physiological demands. What is more difficult, the body has an insulin concentration gradient such that the insulin concentration is decreased in order of: the hepatic portal vein, the liver, the hepatic vein, the aorta and the muscle, but an injection of exogenous insulin does not result in such a concentration gradient, which then causes side effects.
The &bgr;-cells of the Langerhans islets secrete insulin and 11 other materials. Thus, an injection of only insulin can decrease the blood glucose level, but cannot prevent glucopenia and other complications. Since one of the objectives in the treatment of diabetes is to lower the blood glucose level, blood glucose lowering agents are often employed. These lowering agents, however, should not be prescribed for an extended period of time because they result in resistance. Moreover, blood glucose lowering agents were found to cause serious side effects.
Insulin, as mentioned above, is able to lower blood glucose level as well as gives much lower resistance than do blood glucose lowering agents. However, the necessary amount of insulin varies with a patient's conditions so that it is very difficult to timely administrate proper dosage of insulin. Upon improper administration of insulin, anti-insulin antibodies may be formed, making diabetes worse.
For curing diabetes, tissue transplantation has recently been of great interest. For example, the pancreas or Langerhans islets are transplanted into a patient who suffers from diabetes to provide a controlled amount of insulin which is necessary for the patient.
In such cases, however, immune rejection is always problematic and must be considered. When the immune rejection occurs, immune suppressors are administered to the patients. In addition, the number of donors are not sufficient relative to the demand.
The treatment of diabetes by insulin administration was first conducted in 1921 by Banting and Best, but they failed to cure the disease because of a diabetic complication. In 1966, Lillihei of Minnesota University first transplanted a portion of the pancreas into a diabetic patient. By 1977, 57 patients had been subjected to the transplantation. However, less than 10% of them survived for one year or more. Recent development of immune suppressors has increased the survival rate of pancreas or kidney transplant recipients up to 70%. For Langerhans islet transplantation, the survival rate amounts up to 90%.
The first thing into which account is taken is the histocompatibility between donor and recipient. If tissue transplantation is performed between two persons who have different histocompatibility, an immune rejection occurs, leading to the destruction of the transplanted islets at the worst. Generally, 50 donors are needed to discover the necessary histocompatibility for one recipient. If fresh islets are transplanted, a large quantity of fibrous tissues grow out from freshly isolated Langerhans islets and divide and surround them if transplanted, so that the ability of the &bgr;-cells to secrete insulin in response to a stimulus declines greatly.
SUMMARY OF THE INVENTION
In approaching the present invention, the present inventors considered the following:
First, in order for cells or cell groups to proliferate in vitro, they must contain stem cells or progenitor cells therein and be in undifferentiated states. Fortunately, since many stem cells or progenitor cells exist in Langerhans islets, it is highly possible to proliferate undifferentiated Langerhans islets in vitro.
Next, MHC class II antigens, which cause immune rejection, must be absent in the proliferated Langerhans islets and thus, if the blood cells, rich in MHC class II antigens, are eliminated from Langerhans islets, the immune rejection can be greatly reduced in the islet allotransplantation. The longer the islets remain in the culture and proliferate, decreases the immune rejection response.
Finally, fibrous tissues are developed from the crude islets and must be able to be easily removed from the in vitro proliferated islet.
Taking advantage of the above three points, the present inventors tried to proliferate in vitro the Langerhans islets with the aim of preparing them so as to be easily and successfully transplanted in the host for the long term treatment of diabetes.
When the Langerhans islets were grown in a monolayer culture method, they proliferated at a rate of, at most, 80%. However, they poorly secreted insulin so that it was impossible to control the level of the blood glucose. The islets should proliferate at least 5-fold for successful transplantation.
Intensive and thorough research repeated by the present inventors resulted in the discovering that upon in vitro culture in media containing various biochemical materials, the Langerhans islets isolated from rats proliferate at high rates sufficient to be applied for transplantation, release the blood cells from themselves so as to greatly reduce the immune rejection, and function well enough so as to successfully continue to secrete insulin after transplantation according to the present invention.
Therefore, it is an object of the present invention to provide a method for proliferating the Langerhans islets in a suitable state for transplantation, whereby a greatly enhanced treatment effect for diabetes can be brought about.
In accordance with the present invention, there is provided a method for proliferating the Langerhans islets, in which a culture medium is supplemented with radical scavengers, growth factors, a matrix material, nerve growth factor, cell migrating/scattering factors (such as HGF) antinecrosis factors or antiapoptosis factors (such as IGF 1, IGF 2, VeGF) and a cytoskeleton activator (anti-integrin &bgr;1 antibody) at proper culture times and the proliferation is conducted for an extended period of time, so that the Langerhans islets are depleted of the blood cells and also proliferate sufficiently in order to be suitable for transplantation.
The present inventions are directed to a method for in vitro culturing and proliferating isolated Langerhans islets endocrine cells so as to be suitable for transplantation. To initiate the proliferation viable Langerhans islets endocrine cells including cells capable of differentiating into insulin producing cells are collected and placed in a first culturing medium comprising a basal medium supplemen
Afremova Vera
Saucier Sandra E.
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