Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Method of detaching cells – digesting tissue or establishing...
Reexamination Certificate
1998-08-10
2002-12-03
Ketter, James (Department: 1632)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Method of detaching cells, digesting tissue or establishing...
C435S325000, C435S379000, C435S380000, C435S381000
Reexamination Certificate
active
06489164
ABSTRACT:
BACKGROUND
1. Islet Isolation
The insulin producing tissue of the pancreas, the islets of Langerhans, constitutes between about one and two percent of the mass of the pancreas. The isolation of the islets from the remainder of the pancreatic tissue is desirable for laboratory purposes and for transplantation purposes. Transplantation of islets is looked to as a possible treatment for diabetes. Transplanting islets rather than an intact pancreas or pancreatic segments offers several advantages, including the ease of transplantation, the possibility of in vitro treatment to prevent rejection without immunosuppression, the elimination of the pancreatic exocrine function (the secretion of digestive substances from the host tissue), the possibility of cryopreservation of the tissue for subsequent use, and the possibility of xenografts.
In an early method of islet separation, chopped pancreatic fragments are mixed with collagenase and incubated at 37° C. (reviewed in Scharp, World Journal of Surgery 8:143-151 (1984)). The collagenase breaks down or digests the pancreatic tissue, freeing the islets. The collagenase also acts on the islets, so that the islets released early in the process are broken down into single cells. If the process is stopped to protect the islets released early, many islets remain trapped in pancreatic fragments. Therefore only a fraction of the available intact islets are released by this method. This process is particularly ineffective for the isolation of islets from the pancreata of large animals such as humans, dogs, or pigs.
Laboratory islet isolation from rodent pancreata was greatly improved by the discovery that mechanical distension of rodent pancreata increased islet yield by causing mechanical separation of islets from the pancreas tissue. After distension the pancreas is chopped for collagenase digestion. The beneficial effect of this same type of mechanical distension has also been noticed in large animals.
Horaguchi and Merrell, Diabetes 30:455-58 (1982) developed a method for perfusing the dog pancreas with collagenase via the pancreatic duct. Subsequently, a process involving ductal distension of the pancreas with a solution containing collagenase was developed (U.S. Pat. No. 4,868,121; incorporated herein by reference). Inflation or distension of the pancreas is believed to cause some mechanical rupturing of the exocrine tissue or partial separation of the islets from the exocrine tissue, making subsequent collagenase digestion easier.
2. Sonication
Sound waves have been used in the past to aggregate cells and to disrupt cells. For example, ultrasound has recently been used to aggregate cells as a purification procedure. Kilburn, D G, et al., “Enhanced sedimentation of mammalian cells following acoustic aggregation,” Biotechnol.Bioeng. 34:559-62 (1989). In this procedure, cells which are not sufficiently heavy to precipitate out of solution are caused to aggregate by exposure to ultrasound. The aggregates then precipitate out of the solution. This procedure uses a standing wave to aggregate the cells, and the procedure is performed in an echo chamber to create and maintain the standing wave.
Ultrasound has also long been used to disrupt cells. For example, exposure of cells to ultrasound is used to lyse the cells to isolate the nucleic acid contained inside. Crouse, C A, et al., “Extraction of DNA from forensic-type sexual assault specimens using simple, rapid sonication procedures” BioTechniques 15:641-42,644-48 (1993). This procedure uses very concentrated sound waves to disrupt the cell structure. The ultrasonic field is applied at a localized spot, such as a microtip of an emitter.
SUMMARY OF THE INVENTION
The present invention is an improvement on the process for isolating cells, such as islets of Langerhans, which incorporates sonication of the organ, such as the pancreas, as a method for dissociating the cells from other non-desired tissue. The inventors have discovered that sonication of the pancreas in conjunction with collagenase treatment results in a high degree of dissociation of the islet cells that maintain a high degree of integrity. The invention can be applied to the isolation of specific cell types from many different types of organs.
DETAILED DESCRIPTION OF THE INVENTION
The invention is an improved method for the isolation of specific viable cell types from surrounding organ tissue. The technique has specifically been applied to the isolation of islets of Langerhans cells from a pig pancreas as described below in the preferred embodiment. However, the invention is also applicable to the isolation of cell types from other organs and other animals (e.g., cells from organs from transgenic animals, islets from human pancreata). Other potential applications include the isolation of medullary cells from adrenal glands, and the isolation of hepatocytes from liver to be used, for example, as bioartificial liver systems. The organ is harvested and prepared as necessary, such as by removal of undesired segregated tissue or cells. The invention relies on the use of sound waves to accelerate tissue dissociation. The cells released from the dissociated tissue remain intact and viable, allowing separation of desired cells from unwanted tissue. Thus, this invention differs from the prior art wherein cells are either aggregated or disrupted. Implementation of the invention entails three steps.
(1) Treatment of the Organ with Tissue Dissociating Agents to Release Specific Cell Types from the Surrounding Organ Tissue
Tissue dissociating agents will typically include tissue degrading enzymes such as collagenase, trypsin, neutral It protease or dispase, and other proteolytic enzymes, with the preferred embodiment demonstrating the use of collagenase. However, the tissue dissociating agents are not necessarily limited to enzymes. Other examples of tissue dissociating agents are chelating agents for the dissociation of fetal tissue. The length of time required for treatment with dissociating agents will vary depending on the type of the agent, the concentration of agent, and the temperature at which treatment is conducted. Treatment is allowed to proceed until a sufficient amount of tissue has dissociated without causing undue damage to released cells or cellular aggregates. Preferably at least 40%, more preferably at least 75%, and most preferably at least 90% of the tissue is dissociated, while less than 50%, more preferably less than 25%, and most preferably less than 10% of the cells are functionally damaged by treatment with the dissociating agents.
The preferred embodiment below describes the treatment of a pancreas via ductal distension, a method fully described in U.S. Pat. No. 4,868,121. That is a method in which the tissue dissociating effect of the treatment agent is enhanced by is injection of the agent into the pancreas to cause tension that results in some mechanical rupturing of the exocrine tissue or partial separation of the islets from the exocrine tissue. However, the invention is not limited to this form of treatment. Other possible types of the treatment would include chopping the organ smaller into pieces and incubation with a tissue dissociating agent, or use of a dissociating agent with mechanical agitation such as incubation of the organ with marbles in a shaking container. In the preferred embodiment described below, enzyme treatment and sonication occur simultaneously.
(2) Sonication of the Organ Tissue to Further Enhance Dissociation of the Cells of Interest
The sonication step as described in the preferred embodiment was accomplished with a sonicating waterbath. However, it should be appreciated that other types of sonication methods could also be used. These would include acoustic horns, piezo-electric crystals, or any other method of generating stable sound waves, such as with sonication probes. In the preferred embodiment described below, sonication was conducted at about 43 kHz for approximately 20 minutes. Under approximately these same conditions, a sonication frequency of between about 40 kHz to 50 kHz is li
Baird Monty Kahn
Gray Brad
Lamberti Francis
Ketter James
Knobbe Martens Olson & Bear LLP
Li J
Novocell, Inc.
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