Apparatus for isolation of particles, preferably cell clusters

Details Apparatus for isolation of particles, preferably cell clusters Apparatus for isolation of particles, preferably cell clusters

2001-04-30

2004-11-23

Wallenhorst, Maureen M. (Department: 1743)

Chemical apparatus and process disinfecting, deodorizing, preser

Analyzer, structured indicator, or manipulative laboratory...

Means for analyzing liquid or solid sample

C422S105000, C422S105000, C422S105000, C422S063000, C422S067000, C436S043000, C436S054000, C436S177000, C436S180000, C382S128000, C382S133000

Reexamination Certificate

active

06821484

ABSTRACT:

FIELD OF THE INVENTION
The invention relates to the field of isolating cell clusters from a tissue suspension with the special aim of isolating islets of Langerhans.
BACKGROUND OF THE INVENTION
Diabetes is characterized by a defect in the insulin producing beta-cells in the pancreas. The beta-cells are localized in the islets of Langerhans surrounded by exocrine tissue. The responsiveness of the beta-cells to extracellular glucose is essential for a normal glucose homeostasis. There is a great need for isolated islets from both animals and humans for several purposes:
1
. Research on basal physiological and pathophysiological mechanisms in the endocrine pancreas,
2
. Screening and testing of insulinotropic, potential antidiabetic drugs and
3
. allo- and xeno-transplantation of islets. The efforts put into clinical islet transplantation is greatly curtailed by the problems in isolating an adequate islet mass for reducing the diabetic state. The islet harvesting process involves principally two different stages, one is the collagenase digestion of the exocrine- and connective tissue and the second is the separation of the islets from the dispersed tissue suspension. The collagenase digestion technique has been known for many years and involves either injection of collagenase (or another dissociating agent) into the duct system of the pancreas or treatment of small pieces of pancreas with collagenase (Lacy & Kostianovsky, 1967). The tissue disintegrates hereby.
Subsequently to tissue digestion, several different methods can be used for separating the islets from the exocrine tissue. These include, among others,
1
). manual picking of the islets or sucking the islets into a pipette,
2
). serial siewing procedures,
3
). laminar flow channels,
4
). density-gradients,
5
). cell separators. Combinations of these methods have also been reported.
Concerning isolation of islets from smaller animals manual picking of islets is very common. This method is time-consuming and the extended periods used for the isolation may be detrimental for studies on e.g. molecular biology of the islets. In addition, the method involves monotonous and sedentary work. This demanding work is often done by laboratory technicians, students, MDs etc (depending on resources and local tradition).
The time spent on islet isolation can be utilized much more efficiently and/or salary expenses can be reduced greatly by automation. In addition, it is well known that sedentary, monotonous work is related to an increased number of days lost through sickness (e.g. as a cause of pain in the neck- and shoulder-region, headache etc). Other important disadvantages of manual picking of islets are clear: the risk of differences in the handling and selection of the islets by different operators (common to all methods). This can cause a large variation in the measured biological parameters. Clearly, also the time-factor is of importance since it is generally accepted that fast transfer of the islets to an optimal culture-medium prevents damage to the islets.
There has been a general lack of standardization and quality measures of the isolation process, especially within the area of human islet isolation (see (Ricordi, 1991)) despite the fact that in order to improve the isolation process it is imperative to document in detail differences in islet yield and quality.
It is of great importance to develop methods for large-scale and fast automated isolation of islets from both animals and humans and some efforts have been put into this area. It is however evident that none of the previously presented methods have been neither widely accepted nor widely used. This counts especially for the area of animal islet isolation.
In contrast, within the area of human and pig islet isolation the development of automated methods for isolation of islets have greatly improved the outcome. Thus, the well-known automated islet isolator (AII) as developed by Ricordi et al. (Ricordi, Lacy, et al.1988, Ricordi, Finke, et al. 1986) and modifications hereof (Teruya, Idezuki, et al. 1994, Lakey, Warnock, et al. 1997) has enabled clinical and experimental transplantation of islets for treatment of human diabetes. Ricordis method combines collagenase digestion with a device for separation of the islets in the same apparatus. The islets are continuously released from the tissue when it is degraded.
However, the Ricordi method requires additional purification by gradient centrifugation (Ricordi, Finke, et al. 1986, Ricordi Lacy, et al. 1988, Ricordi, Finke, et al. 1988). It may also result in variations in the time the islets are treated with collagenase and thus produce islets with varying quality and characteristics. It may not be advantageous to combine the digestion process with the separation process in the same apparatus.
Several other methods have been developed, e.g. density gradient centrifugation for isolation of human islets and for isolation of islets from rodents dogs, pigs and humans (Marchetti Finke, et al. 1991, Tze, Wong, et al. 1976, Ricordi, Lacy, et al. 1988, Shibata, Ludvigsen, et al. 1976, Buitrago, Gylfe, et al. 1977). A few laboratories combines gradient centrifugation with a cell separator (Lake, Basset, et al. 1989, Olack, Swanson, et al. 1991, Prevost, Rolland, et al. 1995). The use of gradients can be gravely criticized since these may cause osmotic damage to the islet cells (Lake, James, et al. 1986, Lake, Anderson, et al. 1987). Potentially, this could be of great importance for the outcome of islet transplantation. In addition, the purity reported using these methods varies greatly and often is around 75%. It would be of great advantage to increase the purity of the isolate.
Other methods for digestion/separation of islets have been described (see for example (Brunicardi, Suh, et al. 1992, Gray & Baird 1996)), e.g. immunomagnetic isolation (Nandigala, Chen, et al. 1997, Davies, James, et al. 1995) and fluorescence activated cell sorting (FACS) of islets marked with e.g. zinc binding dye (Jindal, McShane, et al. 1994) or neutral red (Gray, Gohde, et al. 1989). Laminar flow channels (Langley. 1993) and filtering of the digested tissue (Sharp, Lacy, et al. 1986) for purification of islets have also been developed. Siewing of tissue preparations is commonly used as a rough, first-line method for getting rid of large undigested tissue pieces.
It is obvious that within the area of animal islet research none of these methods are widely used and the manual islet isolation is still very common and used by most laboratories including those in the pharmaceutical industry. It is clear, therefore, that efforts should be put into facilitation of isolation process since substantial resources are used on this process. In addition, advantages are increased quality of the isolated products and the invention will also reduce inter-operator variation in selection of islets. Improved methods for isolation of rodent islets are useful for the research regarding transplantation and improved methods in this are likely to rub off on the human islet transplantation.
It is an object of the present invention to substantially improve the isolation process. It takes advantage of, and incorporates improvements, of the conventional methods for disintegration of the tissue of interest. It may be used for primary isolation of islets from the surrounding tissue but may also be used for later purification or transfer of islets from one place to another. The primary goal is to provide a fast, reliable apparatus for isolation of pancreatic islets of Langerhans and in the same apparatus implement documentation.
SUMMARY OF THE INVENTION
The invention is concerned with isolation of cell clusters embedded in a tissue suspensign. The apparatus is preferably designed for isolation of pancreatic islets but is applicable to many kinds of cell clusters, single cells, for example spermatozoa, or non biological particles. It takes advantage of conventional methods for disintegration of the primary tissue containing the cell clusters of interest. The principle of the i

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