Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Animal or plant cell
Reexamination Certificate
2001-01-11
2001-11-13
Prats, Francisco (Department: 1651)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Animal or plant cell
C424S424000, C424S492000, C514S772000, C514S774000, C435S182000, C623S011110
Reexamination Certificate
active
06315994
ABSTRACT:
FIELD OF INVENTION
The present invention relates to a cell culture medium and matrix composition for preserving cell viability as well as gene expression and specialized tissue function. The present invention also relates to a matrix capable of sustaining cell viability after injection of hormone secreting cellular moieties into living tissue.
BACKGROUND OF THE INVENTION
New methods for treating insulin-dependant diabetes mellitus are presently being sought. At the present time, diabetes patients test their blood sugar levels and inject insulin when necessary. Although it is possible to transplant a pancreas from one human to another, the survival rate for this procedure is only 40% at one year following surgery. Researchers have used isolated pancreatic islets in transplantation approaches in attempts to find a viable long term treatment of diabetes.
The islets of Langerhans are clusters of differentiated cells sharing a common precursor. Found in the pancreas of mammals, islets taken together can be considered as a single endocrine organ. The islets occupy about 7% of the human pancreas which also contains the exocrine acinar tissue. The composition of cells in the islets differs depending on the location of the islet in the pancreas. Central to each islet is a core of insulin secreting beta cells. Surrounding the beta cells are somatostatin secreting delta cells, glucagon secreting alpha cells and pancreatic polypeptide containing f cells. Alpha cells tend to be concentrated in the tail and the body of the pancreas whereas, the f cells are concentrated in the head. This distribution corresponds to the embryonic origin of alpha and f cells from dorsal and ventral primordium of the pancreas.
Pancreatic beta cells are the only cells in which the insulin gene is expressed and, therefore, are the sole source of metabolic insulin in vertebrates. Insulin is necessary in maintaining glucose homeostasis and plays a role in the normal processing of proteins and fats. Insulin release can be inhibited by low levels of somatostatin and stimulated by glucagon. Without sufficient insulin to metabolize glucose, hyperglycemia occurs. Insulin-dependant diabetes mellitus is a direct result of nonfunctional islets, specifically beta cells.
Among the major obstacles in islet transplantation research is an inability to induce proliferation and to keep islets viable over time. Researchers have encountered many obstacles in attempting to cure diabetes resulting from the loss of islet function. For transplantation, it is necessary to preserve islet viability as well as gene expression and secretory function.
Pancreatic islets do not grow readily in primary cultures. However, these endocrine cells have been grown with difficulty as monolayers. This difficulty of long-term culture has not only hindered the laboratory research for such islets, but it has also hindered attempts to carry out physiological and even clinical studies with such islets. Therefore, there is needed a medium for the long-term proliferation of islets. A medium for the long-term survival of cells is additionally needed for other cell types.
Additionally, current methods of transplantation must suppress immune response by the host organism that may lead to rejection of the transplanted cells and loss of islet function. Thus, there is also a need in the art for a simple, non-invasive method of introducing hormone secreting cellular moieties, such as insulin secreting pancreatic islets, into a hormone deficient organism without requiring general immunosuppressive agents.
SUMMARY OF THE INVENTION
A cell culture medium to promote the proliferation and long-term survival of cells is provided. The cell culture medium includes elevated levels of polar amino acids. The addition of polar amino acids to the medium enhances cell proliferation and maintains cell viability for sustained periods of time.
Additionally, a hydrogel matrix for the long-term proliferation of cells is provided. The matrix includes elevated levels of polar amino acids. The matrix of the present invention may be used as a carrier for direct injection of cells into a host organism without significant loss of cell viability or function. Additionally, the matrix acts to shield the cells from the immune system of the host organism.
Also provided are transplants capable of long-term functioning in a host. In particular, insulin secreting transplants comprising islet cells and acinar cells are provided. The transplants of the present invention include the matrix and allows coexistence of islet and acinar cells with improved insulin pulsatility.
A cell culture medium for long term storage and proliferation of cells is provided. The cell culture medium includes an effective amount of polar amino acids. Preferred polar amino acids are selected from, but not limited to, the group consisting of arginine, lysine, histidine, glutamic acid, and aspartic acid. The effective amount of polar amino acids is preferably about 5 to about 150 mM and most preferably about 10 to about 64 mM. In one embodiment, the polar amino acids comprise about 2 to about 60 mM of arginine and about 2 to about 60 mM of L-glutamic acid. The cells cultured in the medium may be selected from a group consisting of lung cells, liver cells, kidney cells, thymus cells, thyroid cells, heart cells, brain cells, pancreatic islet cells, pancreatic acinar cells, and mixtures thereof.
A hydrogel matrix for long term storage and proliferation of cellular tissue is also provided, the matrix comprising about 0.01 to about 40 mM of gelatin and an effective amount of polar amino acids. The effective amount of polar amino acids is preferably from about 3 to about 150 mM and most preferably about 10 to about 65 mM. In one embodiment, the polar amino acids are selected from the group consisting of arginine, glutamic acid, lysine or mixtures thereof. Preferably, the hydrogel matrix includes about 2 to about 60 mM of L-glutamic acid, about 1.5 to about 10 mM of L-lysine and about 1 to about 40 mM of arginine.
A method of maintaining, cell viability and functioning during storage is provided wherein the cells are imbedded in the hydrogel matrix of the present invention. The matrix protects cells during storage, including frozen storage.
A method of maintaining viability and functioning of a transplant cell after introduction into a host organism is also provided. The method includes the steps of embedding the cells in the hydrogel matrix described above and injecting the embedded cells into the host organism. While the majority of the matrix liquifies and is absorbed by the host, polar moieties of the matrix attach to cell surface polar moieties, thus obscuring cell surface immune recognition proteins. Advantageously, the hydrogel matrix may be injected into a transplant site prior to injection of the cells to encourage vascularization. The encapsulated cells may be isolated from a different species than the host organism.
The matrix of the present invention may also be used to stimulate vascularization at a site in a host organism to treat conditions benefitted from an increased supply of blood. The method includes contacting the site with the matrix of the present invention wherein the matrix comprises an effective amount of polar amino acids.
A transplant for implanting in a host organism is also provided. The transplant comprises cells having outer surfaces encapsulated by a matrix comprising an effective amount of polar amino acids. The effective amount of polar amino acids may be about 3 to about 150 mM. These polar amino acids serve to enhance bonding of other polar moieties and further obscure immune recognition proteins in a host subject. Thus, cells embedded in this enhanced hydrogel matrix substantially escape host immune destruction.
A method for increasing insulin production in a transplant is also provided. Insulin production may be increased in a transplant by providing a mixture of acinar cells and islet cells and encapsulating that mixture in a matrix comprising an effective amount of polar amino acids to form a
Klann Richard Chris
Usala Anton-Lewis
Alston & Bird LLP
Ozga Brett
Prats Francisco
LandOfFree
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