Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Method of regulating cell metabolism or physiology
Reexamination Certificate
2001-11-27
2004-12-21
Lankford, Jr., Leon B. (Department: 1651)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Method of regulating cell metabolism or physiology
C435S378000, C435S379000, C435S380000, C435S381000
Reexamination Certificate
active
06833270
ABSTRACT:
FIELD OF INVENTION
This invention relates generally to an apparatus and method for isolating cells from organs.
BACKGROUND OF INVENTION
Scientists are currently researching possible applications for isolated cells from parent organs, such as the liver, spleen, kidney, adrenal, and pancreas. Some research that has been conducted on the clinical application of isolated cells has involved groups of cells called the Islets of Langerhans that have been isolated from the pancreas. An application for the Islet of Langerhans cells is as a treatment for diabetic patients. Patients with diabetes have Islets of Langerhans that do not function properly, and therefore, do not produce enough insulin. Some clinical research is aimed at developing a procedure for transplanting functioning Islets of Langerhans into diabetic patients to restore the insulin producing ability of the pancreas. Clinical research of such requires isolated Islet of Langerhans cells, but these cells must be isolated while still viable. Viable isolated cells are mostly obtained from organs of the very recently deceased. The apparatus and method for isolating the cells should be able to extract isolated cells with as little damage to the cells as possible.
Many different methods and approaches have been attempted to isolate individual cells from their respective parent organs. Prior methods have produced isolated cells with some cell destruction. This cell destruction can result from the relatively severe mechanical stimulation that is used to isolate cells from an organ.
One method that attempts to overcome the loss of damaged cells due to relatively severe mechanical stress is described in U.S. Pat. No. 5,079,160, to Lacy, et al. The method disclosed by Lacy, et al. comprises the steps of: placing an organ or a piece of an organ in a digestion chamber along with marble agitators; distending the organ or a piece of the organ with physiologically compatible medium containing a protease; continuously recirculating that medium; and separating the isolated cells. The marble agitators greatly increase the amount of undamaged cells obtained through isolation without reducing the quality of the isolated cells obtained by gently agitating the organ. Moreover, the marbles are an appropriate size, weight, and density for obtaining beneficial results as compared to other agitators of varying size, weight, and density which can cause severe mechanical disruption of the organ tissue resulting in some cells being destroyed.
Although the Lacy, et al. method produces isolated cells in relatively high concentrations, the use of marble agitators has significant disadvantages. The marble agitators do not have a high level of hardness. Also, the marble agitators can be brittle or become brittle and can break, fracture, or chip during the isolation of cells. Furthermore, due to stringent sterilization standards, the marble agitators must undergo relatively stressful sterilization procedures in which the marble agitators may become brittle, break, fracture, or chip.
SUMMARY OF THE INVENTION
The present invention relates to an apparatus for extracting cells from organs. The apparatus includes a digestion chamber that contains a physiologically compatible medium with at least one protease. The digestion chamber has at least one inlet and at least one outlet, and has a separator means for retaining the organ and permitting the cells and the physiologically compatible medium to exit the outlet. The apparatus also has at least one agitation member in the digestion chamber, and the agitation members have an interior with at least one void.
In one embodiment, the agitation members can include a non-corrosive metal and a substantially smooth, continuous exterior surface, and can be substantially spherical. The agitation members can also have an interior with one centrally located substantially spherical void. Additionally, the agitation members can have a density of about 3.0-4.0 g/cm
3
.
Moreover, the agitation members can have a density of about 3.5 g/cm
3
.
Furthermore, the present invention relates to agitation members for the digestion chamber of an apparatus for extracting cells from organs. The agitation members have an interior with at least one void. Additionally, the agitation members can have the characteristics listed above.
The present invention also relates to a method for extracting cells from an organ. The method includes the steps of: providing a physiologically compatible medium with at least one protease; providing a digestion chamber, the chamber having at least one inlet and at least one outlet, and a separator for retaining the organ and for permitting the cells and the physiologically compatible medium to exit the outlet; providing at least one agitation member in the digestion chamber, the agitation members having an interior with at least one void; flowing the physiologically compatible medium through the digestion chamber; moving the agitation members within the digestion chamber, whereby the agitation members will agitate the organ to facilitate release of the cells; and collecting the cells. In regard to the step of moving the agitation members, the method can further include a step of moving the digestion chamber so as to move the agitation members within the digestion chamber. The agitation members can also have a density of 3.0-4.0 g/cm
3
, and can have a density of 3.5 g/cm
3
.
The invention can also relate to a method for extracting cells from an organ in which the organ is a pancreas and the cells are Islet of Langerhans. Also, the protease in the physiologically compatible medium can be collagenase. In one aspect, the physiologically compatible medium can be heated prior to entering the digestion chamber to a temperature selected to maximize the effectiveness of the protease. Therefore, in one aspect, the heating can heat the physiologically compatible medium to a temperature of 24° C.-40° C. In yet another aspect, the physiologically compatible medium can be heated to a temperature of 37° C. The physiologically compatible medium can also be cooled following exit from the outlet of the digestion chamber to a temperature between 4° C.-20° C. Additionally, prior to the step of collecting the cells, the method can include a step of detecting the cells in the physiologically compatible medium. The method can also include the step of removing the physiologically compatible medium containing the cells and adding additional physiologically compatible medium without heating prior to entering the digestion chamber.
REFERENCES:
patent: 4017361 (1977-04-01), Febvre
patent: 5079160 (1992-01-01), Lacy et al.
Poo Ramon E.
Ricordi Camillo
Akerman & Senterfitt
Biorep Technologies, Inc.
Lankford , Jr. Leon B.
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