CELL SEPARATION INVENTION

Abstract: An aqueous composition and a method of use are disclosed for the separation of different living cells, such as pancreatic islet and acinar cells. The composition comprises a water soluble, metabolically inert substance to control density, osmolarity and pH; a physiological cold storage solution containing viscosity modifiers to increase the viscosity and osmotically active, impermeant agents to suppress cold-induced edema; all in a medium capable of maintaining viable cells. The solutions are combined to provide a first solution with a density greater than both cell types in their natural state but less than the second cell type after incubation in said solution; and a second solution with a density less than that of both cell types. Separation is achieved by allowing the system to stand or by centrifugation.

Description: DESCRIPTION Cell Separation Invention This is a continuation-in-part of co-pending applica¬ tion U.S. Serial No. 07/410,091. Field of the Invention: This invention relates to the fields of cell biology and medicine, and more particularly, to the field of mammalian cell separation of, particularly for insulin- producing cells. Art Background: Isolation and purification of a specific cell population is an important issue in many areas of cell biology. Several methods of purification of cells have been used over the years, including centrifugal separation based upon size or density, cloning and immunological (antibody) recognition and separation, among others. Over the years, many different compounds have been used to form density gradients to enable particles to be separated according to their size and/or buoyant density. Some common materials for this purpose include ficoll, percoll, cesium chloride and dextran. While these materials may have been used for the separation of cells, they have certain drawbacks which make them not particu¬ larly well-suited for this task. These drawbacks are due in part to sensitivity of the cells to high osmotic or ionic stress, sensitivity to the toxic compounds used, and stability of the separation compounds to autoclaving or other sterilization techniques. When cells are separated using centrifugation, they may be separated by size or buoyant density, or to a minor extent, by charge or other related external surface char- acteristics. With respect to separation processes relying upon differences in size, when particles in solution are subjected to a centrifugal field, they move in the direc¬ tion of the force applied, and in general, larger parti¬ cles will move faster than smaller particles. Therefore, when cells differ greatly in size, a reasonable purifica- tion may be obtained by low speed centrifugation in a suitable medium. In cell suspensions containing a mixed population of cells, cells of similar sizes may have different buoyant densities, whereas cells of different sizes may have the same densities. Cells having different buoyancy charac¬ teristics may be separated on a density gradients, such as continuous gradients or discrete step (discontinuous) gradients. In either case, the principle is that the cells will migrate through the gradient medium until they reach a point where the density of the medium equals the density of the cells, in the case of a continuous gradi¬ ent, or where the cells are sandwiched at the interface in between a medium having a lower density and a medium having a greater density than the cells, in the case of a discontinuous gradient. This method results in the cells being disposed in discrete bands within the medium. An example of the need for rapid isolation and purification of cells is the desirability of acquiring large quantities of pure insulin producing cells from a pancreas for purposes of transplanting them into a dia¬ betic patient. The pancreas is the organ responsible for insulin production. Specifically, insulin is produced and regulated by areas of the pancreas known as the Islets of Langerhans, referred to herein as islet cells or islets, which are the endocrine cells of the pancreas. Such cells comprise a small percentage of the pancreas (around 2%) . The major cellular component of the pancreas consists of exocrine tissue including acinar and ductal cells, and it has been shown to be a formidable task to purify the islet cells from the acinar cells. Gray and Morris, Transplan¬ tation (1987) 42:321. Islet cell isolation and purification is currently being performed by density gradient separation based upon the principle of density differences between the isolated islets and the acinar cells. Generally the dispersed pan- creatic preparation is placed in a discontinuous density gradient solution containing ficoll, percoll or dextran, all well-known density gradient materials. There are several disadvantages to prior art methods of islet cell isolation and preparation utilizing such materials. For one, the method is cumbersome, time consuming and labor intensive since several gradients are required to be layered and the islets must be carefully removed from within these multiple gradients. Another disadvantage is that the method yields inconsistent results since the density of the islet cells and acinar cells may change during the process as a result of edema in the cells caused by the materials used in the separation. Another serious disadvantage of the prior art methods is that the cells are subjected for substantial periods of time to the gradient material which may be toxic, and is at least detrimental to the viability of the cells. This is par¬ ticularly the case because the currently used gradient solutions such as ficoll, percoll and dextran are not phy¬ siological solutions thus causing both osmotic and ionic stresses on the cells. Ficoll is known to be toxic to cells as well as mutagenic. Percoll also causes cellular damage. Dextran in the concentrations used in gradient separation may cause cellular damage as a result of the osmotic stresses applied to the cells. Furthermore, none of the gradient separation materials are capable of pre¬ serving cells for any substantial periods of time. Summary of the Invention The present invention comprises a cell separation material useful in the separation and purification of cells, particularly islet cells, and a method of cell separation using the invented material. The material has properties and characteristics of a physiological solution and therefore is not harmful and is even somewhat benefi¬ cial to the cells. Moreover, it has cell preservation properties, being derived in part from a tissue preserva- tion solution. The invented material is capable of separating two mixed types of cells, such as acinar and islet cells, without being entirely dependent upon the naturally occurring densities of the respective cells. Moreover, the multiple gradients required in the prior art separa¬ tion techniques are not required in the present invention in that an initially two phase aqueous separation system is capable of performing the desired separation. The composition of this invention is made of heat stable material. This facilitates sterilization by allowing autoclaving. The present invention is a composition comprising two aqueous phases, as well as a continuous gradient which develops between the two phases, of solutions useful in the isolation and purification of two types of cells from a mixture. In particular, this invention is useful in separating islet cells from a pancreatic tissue digestate containing acinar cells in addition to the islets. The composition permits high viability of at least one type of cells, such as the islet cells, especially under cold conditions (such as 4°C), and permits these cells to be fully biologically active after the separation. The present invention also comprises a method of isolating and purifying islet cells using the composition. Brief Description of the Drawings FIGURE 1 is a schematic view of the method of the present invention. FIGURE 2 is a graph demonstrating the viability and effectiveness of islet cells obtained by the invented method, which cells were obtained from canine pancreatic tissue and implanted into diabetic rats. FIGURE 3 is a graph demonstrating the viability and effectiveness of islet cells obtained by the invented method, which cells were obtained from rat pancreatic tissue, encapsulated in an alginate capsule and implanted into diabetic rats. This graph shows that the islet cells implanted in the rat kidneys were functional for at least ten weeks. FIGURE 4 is a bar graph demonstrating in vitro that the islet cells recovered from the experiment discussed above in connection with Figure 3 after 75 days of implan¬ tation were still reactive to glucose by producing insulin. FIGURE 5 is a graph also demonstrating the effect¬ iveness of the encapsulated islet cells of the experiments of Figures 3 and 4. in vitro by constant perfusion of the encapsulated cells with first 60 mg% and then 300 mg% glu¬ cose and then 60 mg% glucose again and the insulin release is measured. FIGURE 6 is a photograph of a 50 ml centrifuge tube containing rat pancreatic tissue and showing the separa¬ tion of the acinar cells which are pelleted at the bottom of the tube and the islet cells which banded at the interface near the center. FIGURE 7 is a lOOx microscope photograph of a sample taken from the center (interface) of the tube of FIGURE 6 showing only islet cells. FIGURE 8 is a lOOx microscope photograph of a sample taken from the bottom of the tube of FIGURE 6 showing only acinar cells. Detailed Description of the Invention Both initial phases of the composition can be made from the same solution, but with different densities. This solution is a combination of a density gradient component and an organ preservation solution comprising an osmolarity adjusting agent and a viscosity modifier. The density gradient component is a water soluble, metabolically inert substance which maintains density, osmolarity and pH in the presence of cells, and is useful in gradient separation. An example is 5-(N-2,3-dihy- droxypropylacetamido) 2,4,5-tri-iodo-N,N'-bis(2,3-dihy- droxypropyl)isophthalamide sold under the trademark Nycodenz ® by Nycomed AS of Norway and Robbins Scientific (Mountain View, California) . This material has a mole¬ cular weight of 821 and a density of 2.1 g/ml. It is very soluble in water and its osmolarity is near physiological. Unlike ficoll and percoll, 5-(N-2,3-dihydroxypropylacet- amido) 2,4,5-tri-iodo-N,N'-bis(2,3 dihydroxypropyl)iso¬ phthalamide is non-toxic to cells and is heat stable, even in solution, making it autoclavable for purposes of sterilization. Another water soluble, metabolically inert substance which may be used in the present invention is a tri-iodinated benzamide derivative of glucose sold under the trademark Metrizamide ® , also available from Nycomed AS of Norway and Robbins Scientific (Mountain View, California) . The 5-(N-2,3-dihydroxypropylacetamido) 2,4,5-tri- iodo-N,N'-bis(2,3-dihydroxypropyl)isophthalamide or other water soluble, metabolically inert material is dissolved in solution. The aqueous solution in which the water soluble, metabolically inert material is dissolved has a viscosity modifier comprising one or more colloids or polymers so that the solution has a viscosity in the range of approximately 1.5 to 3.2 mPas. Such viscosity modi¬ fiers include colloids such as hydroxyethyl starch or serum albumin, or polymers such as polyethylene glycol, dextran and alginate. Alginate can be used in concentra¬ tions ranging from 0 to 2.5% of the solution to provide the required viscosity of the solution. The solutions can be isotonic to the cells to be separated. However, in one aspect of the invention, the second of the two types of cells in the mixture to be separated is more permeable than the first. In this case. if the solutions are somewhat hypertonic, this more permeable second cell type will increase in density with incubation in the hypertonic solution. This aids separa¬ tion of the two cell types by increasing the difference in density of the two cell types. For example, when the first cell type is islet cells and the second cell type is acinar cells, the acinar cells can preferentially be made more permeable by directing collagenase enzymes to acinar cells via ductal injection of the enzymes. The acinar cells then increase in density with incubation, aiding in cell separation. The islet cells, on the other hand, are preserved in their natural state and are kept viable by this procedure. To effect separation of the cells after incubation, the density of the solution for use in the lower phase of the gradient is made to be heavier than the density of the second type of cells in their natural state, but lighter than the density of the second cell type after they incubate in this solution. When the solution is made in the range of approxi- mately 290 - 320 mOsm, which is at or very close to the physiological range of osmolarity for most cells, the yield of the desired, less permeable cells is optimized. At an osmolarity of somewhat higher levels, i.e. between approximately 320 and 550 mOsm, purity of the desired less permeable cells is optimized. Therefore, in order to achieve a reasonable level of both purity and yield, a range of approximately 320 to 440 mOsm is preferred. Osmotically active impermeants are osmolarity enhancing compounds which do not permeate into the cells. Such materials are useful in providing a physiological environment for cells without causing cell edema to the preferred cell type, whereas, traditional gradient separ¬ ation materials contribute to cell edema in all cells exposed to those materials. Osmotically active agents which are beneficial in the invented solution include sugars such as glucose, mannitol, sucrose, anions such as phosphate, sulf te, glycerophosphate, citrate and gluco- nate, and compounds which specifically prevent or suppress cold-induced edema such as lactobionate acid or raffinose. Organ preservation solutions which could be used are Eurocollins and UW solutions. UW solution, in particular, has been reported as an organ preservation solution for liver and pancreas transplantation (Lancet 7:617-619, 1988) obtained from DuPont Critical Care (Waukegan, Illinois) . UW solution is comprised of the following components: Ingredient Concentration/1iter K lactobionate 100 mmol NaKH 2 P0 4 25 mmol Adenosine 5 mmol MgS0 4 5 mmol Glutathione 3 mmol Raffinose 30 mmol Allopurinol 1 mmol Insulin 100 U Trimethoprim/sulphamethozazole 8 mg/40 mg Modified hydroxyethyl starch 50 g Na 30 mmol/1 K 120 mmol/1 pH 7.4 320-330 mmol/1 The first, or lower, solution has a density somewhat greater than the density of both cell types in their natural state, in the range of 1.097 to 1.126 g/ml for islet-acinar cell separation. This density will prevent the preferred cells, such as the islets, from entering the lower phase of the gradient. The cells of the second type, which are somewhat permeable to the solute in which they are incubating, as well as cells which are no longer viable due to the preferential degradation through the use of enzymes such as collagenase, become more dense than in their natural state through uptake of the solute. These cells will then enter the lower phase. The lower phase should be at a density at or slightly lighter than the heavier cells become after incubation, so that the heavier cells will in fact fall through the lower phase and settle at or near the bottom of the container. One means to determine the appropriate density for the lower layer is to incubate a crude prep of the heavier cells in various densities of the solution that forms the lower layer. Observing these incubating cells after a period of time long enough for the cells to change in density will show which density is appropriate for the lower phase of the gradient. The upper phase of the two phase aqueous solution can be made of the same solution as the lower phase, but at a different density. Alternatively, the upper phase can be a physiological solution containing nutrients, such as a culture medium like RPMI, Hank's solution, MEM, Eagles medium, or the like. When the upper phase is made of a different density version of the same solution as the lower phase, a small, third layer of physiological solu¬ tion is preferably added as an uppermost layer to trap any fat cells contained in the mixed population, such as from tissue digestate. The density of the upper phase is less than that of both cell types, and for islet-acinar separation is less than 1.050, preferable less than 1.008, and most prefer¬ ably around 1.004 g/ml.. The greater the difference between the densities of the initial two phases of the gradient, the wider the continuous gradient is that forms during separation. This wider continuous gradient region will allow better separation of the two cell types. As noted above, the subject invention also comprises a method of isolating and purifying one population of cells, such as islet cells, using the invented two phase aqueous solution. The invented method comprises gener¬ ally the steps of (1) obtaining a mixture of cells, such as a pancreatic digestate, preferably in a manner which preferentially slightly damages one population of cells, such as acinar cells making them more permeable, (2) combining the mixture of cells in the bottom (first) solution in a container such as a centrifuge tube, (3) adding the second solution to the container, with an optional third uppermost layer, as described previously, and (4) allowing separation, either by standing or centri- fuging at a low speed for a sufficient time and speed to obtain separation of the two cell types, such as acinar from islet cells, with the denser, or second, cell type, such as damaged or non-viable acinar cells, migrating to or near the bottom of the tube and the lighter, or first, cell type, such as islet cells, collecting at or near the interface between the two solutions. During this separa- tion a short, continuous gradient forms between the two discreet phases, providing a range of densities inter¬ mediate between those of the two original phases. The lighter or first type of cells, such as the islet cells, collect at the density within this continuous gradient corresponding to their internal density. Finally, (5) cells of the first, or desired type, such as islet cells, are removed from the gradient where they have formed a band. With an islet-acinar mix, the purity of the islet cells is very high, and may be in the range of 85% to 95%, depending on the preparation and technique. For islet-acinar separation, the pancreatic tissue may be obtained from any pancreas containing animal, such as pig, cow, dog, rat, monkey or human. The pancreatic tissue is obtained from the donor animal and then digested into a cell suspension by any of various methods known in the art such as the method described in Warnock, et al, Transplantation. 45:957-963, No. 5, May, 1988. These procedures utilize ductal injection of collagenase to preferentially damage acinar cells, which are present surrounding the ducts. The pancreatic tissue is prefer¬ ably maintained at 4 β C or similar cold temperature during the entire isolation and purification process to ensure that the enzymes contained in the acinar cells do not have an opportunity to lyse the islet cells. The mixed population of cells, such as pancreatic tissue digestate so obtained, is suspended in the first solution which is preferably 5-(N-2,3-dihydroxypropyl- aceta ido) 2,4,5-tri-iodo-N,N'-bis(2,3-dihydroxypropyl)- isophthalamide in UW solution. The mixed population such as the pancreatic tissue is first suspended in about 1 to 5 ml of physiological Hank's or similar aqueous material and then mixed into a volume of the first solution to create the appropriate density. Any container appropriate for the separation of gradient layers can be used. If one rat pancreas is used, about 15 ml of the first solution can be used in a 50 ml centrifuge tube, and if one canine pancreas is used about 150 ml of the first solution can be used in a 250 ml tube. The second solution, preferably RPMI, is then added to the container. At least approximately 1/4 of the volume of the first solution is layered over the first solution. The gradient can then be allowed to incubate at lg for a period of at least 30 minutes, or until the heavier cells, e.g. the acinar cells, separate and fall to or near the bottom of the container. Alternatively, the container can be centrifuged, preferably for 15 minutes at 4"C at a speed sufficient to create a centrifugal force that does not damage the first, or desired cell type, preferably at a speed sufficient to create a centrifugal force at the interface between the first solution and second solution up to around 180 g. The lighter cells, e.g. the islet cells, are then removed from at or near the interface. The lighter cells thus obtained are a rela¬ tively pure culture, being free from the heavier cells, and being relatively viable and healthy. Figure 1 shows a general schematic step diagram of the invented method. The term PIPS stands for the physio¬ logical islet purification solution, or the first solution of the present invention. In the pancreatic digestate, the dark circles represent the islet cells and the smaller irregularly shaped objects are the acinar cells in the mixed population of the digestate. Figures 6, 7 and 8 are several photographs illus- trating the method of the present invention. Figure 6 is a photograph of a 50 ml tube after centrifugation con¬ taining the two phase aqueous solutions with the islet cells disposed at the interface and the acinar cells disposed in the bottom of the tube. Figure 7 is a photo- graph of a microscopic view of the cells taken from at or near the interface showing that only the islet cells are present at the interface. Figure 8 is a photograph of a microscopic view of the cells taken from the bottom of the tube showing that only acinar cells are present there. Several experiments were ' performed to demonstrate the effectiveness of the subject invention. Experiment 1 Comparative In Vitro Viability Islet cells were isolated, hand-picked and separated into three groups. Equal numbers of cells were incubated for 30 minutes at 4 β C into three different solutions, RPMI, PIPS and ficoll. The PIPS and ficoll were both dissolved into UW solution. Both populations were removed from their respective incubation solutions and placed in a physiological solution to measure the cell function in terms of insulin production. The cells were incubated in specified glucose solutions of 60 mg%, which is physio¬ logical base line, and 300 mg% which is elevated, and which should induce full cellular production of insulin. The cells were then returned to the 60 mg physiological basal level of glucose. As can be seen from the following Table 1, the cells incubated in PIPS produced over twice the insulin (10.4 compared with 4.6) as the ficoll incubated cells. The numbers in parenthesis are the stim- ulation index, that is, the ratios between the insulin production compared with the baseline value at the first 60 mg% incubation. Experiment 2 Transplantation of Purified Human Islets Human islets were isolated in accordance with this invention. A nude rat was transplanted with 4000 human islets in a blood clot under the renal capsule. Four weeks post-transplant, the rat was anesthetized and can- nulated for systematic blood sampling. Heparinized saline (10 units/ml) was used in the cannula to replace blood volume and 0.3 to 0.4 ml of blood was removed at time 0, 5, 15, 30, 45 and 60 minutes after glucose injection of 3 mg/ml of body weight. Plasma was frozen for subsequent RIA of insulin and blood glucose measurements in accord¬ ance with procedures known in the art. Blood was analyzed in duplicate. The results are set forth in Table 2 below. The table shows the insulin and blood glucose concentra- tions from the experiment at the specified time intervals. As can be seen, high levels of insulin release occur in response to glucose stimulation, demonstrating excellent viability of islets isolated in the invented solution utilizing the invented method. Experiment 3 Transplantation of Purified Dog Islets Islet cells obtained from canine pancreas in accord¬ ance with the present invention were encapsulated in accordance with procedures described by Lim and Sun in order to prevent immunological rejection due to histocom- patibility differences resulting from the use of the cells in a different species. The encapsulated islets were then transplanted into a diabetic Lewis rat. The blood glucose levels were then monitored on a daily basis. As shown by Figure 2, the initial blood glucose level was greater than 450 mg%. After transplantation of the encapsulated islets, the level dropped to approximately 100 mg% and remained at that level for the 6 days for the experiment, demonstrating in vivo viability of the canine islets isolated using the present invention. Experiment 4 Longterm In Vivo Viability Figures 3, 4, and 5 show the result of an experiment wherein rat islet cells purified in accordance with the present invention. The islet cells were encapsulated as discussed above and implanted into diabetic Lewis rats for up to 75 days. Figure 3 shows that the islet cells func¬ tioned for the entire length of the experiment in that the serum glucose levels dropped from over 450 mg% prior to implantation to 100 - 150 mg% and remained at that level for the 75 days until the experiment was terminated. The encapsulated cells were then removed from the diabetic rat into which they were transplanted and the function of the islet cells was then tested in vitro. The cells were exposed to 60 mg% glucose, 300 mg% glucose and then 60 mg% glucose and the production of insulin was mea¬ sured. These results were compared with islets that were tested prior to transplantation. As can be seen from the bar graph, the 300 mg% incubation caused the islets to release substantial amounts of insulin and the post trans¬ planted and pre-transplanted cells functioned nearly the same, and in fact the post transplantation cells functioned slightly better. In a similar experiment, the results of which are shown in Figure 5, the post-transplanted islets were per¬ fused with a solution containing varying concentrations of glucose. The cells effectively produced insulin after exposure to 300 mg% glucose and the production of insulin decreased immediately after the glucose concentration in the perfusion medium was decreased to 60 mg% again. It will be obvious to a person of ordinary skill in the art that the present invention is not limited in its application to the specific materials and methods described as the preferred embodiments herein. The only limitations of the present invention are set forth in the claims appended hereto and any equivalents thereof.

Claims: Claims 1. An aqueous composition, initially present as a two-phase gradient, useful in the isolation and purifica¬ tion of two cell types having different densities, wherein a first cell type has a higher density than a second cell type after incubation in the first or lower solution, said composition comprising: A) a first, or lower solution having a density no higher than the density of the first cell type, said first solution comprising: a water soluble, metabolically inert substance which maintains density, osmolarity and pH in the presence of cells; and a physio¬ logical cold storage solution comprising viscosity modifiers to increase the viscosity of said first solution and osmotically active impermeant agents for suppressing cold-induced cellular edema B) and a second, or upper solution having a density less than the second cell type, said second solution selected from the group comprising: 1) a physiological solution capable of maintaining viable cells; and 2) a water soluble, metabolically inert substance which maintains density, osmolarity and pH in the presence of cells; and a physio¬ logical cold storage solution comprising viscosity modifiers to increase the viscosity of said first solution and osmotically active impermeant agents for suppressing cold-induced cellular edema, overlayed with a physiological solution capable of maintaining viable cells. 2. The composition of claim 1 wherein the physio¬ logical solution capable of maintaining viable cells is selected from Hank's solution, and tissue culture media. 3. The composition of claim 1 wherein the water soluble, metabolically inert substance is selected from the group 5-(N-2,3-dihydroxypropylacetamido) 2,4,5-tri- iodo-N,N•-bis(2,3-dihydroxypropyl)isophthalamide and Metrizamide ® . 4. The composition of claim 1 wherein the physio¬ logical cold storage solution is selected from the group UW solution and Eurocollins. 5. The compositon of claim 1 wherein the first cell type is islet cells and the second cell type is acinar cells. 6. The composition of claim 5 wherein the density of the first, or lower solution is between 1.097 and 1.126 g/ml and the density of the second or upper solution is less than 1.050 g/ml. 7. The composition, of claim 1 wherein the first and second solutions are slightly hypertonic to both cell types. 8. An aqueous composition, initially present as a two-phase gradient, useful in the isolation and purifica¬ tion of islet cells from acinar cells wherein the acinar cells have achieved a higher density than the islet cells through incubation in a first, or lower solution, said composition comprising: A) said first, or lower solution having a density between 1.097 and 1.126 g/ml, said first solution comprising: a water soluble, metabolically inert substance which maintains density, osmolarity and pH in the presence of cells; and a physio¬ logical cold storage solution comprising viscos¬ ity modifiers to increase the viscosity of said first solution and osmotically active impermeant agents for suppressing cold-induced cellular edema, B) and a second, or upper solution having a density less than 1.050, said second solution selected from the group comprising: 1) a physiological solution capable of maintaining viable cells; and 2) a water soluble, metabolically inert substance which maintains density, osmolarity and pH in the presence of cells; and a physio¬ logical cold storage solution comprising viscos¬ ity modifiers to increase the viscosity of said first solution and osmotically active impermeant agents for suppressing cold-induced cellular edema, overlayed with a physiological solution capable of maintaining viable cells. 9. The composition of claim 8 wherein the islet and acinar eels are added to the first, or lower solution. 10. The composition of claim 8 wherein the physio¬ logical solution capable of maintaining viable cells is selected from Hank's solution, and tissue culture media. 11. The composition of claim 8 wherein the water soluble, metabolically inert substance is selected from the group 5-(N-2,3-dihydroxypropylacetamido) 2,4,5-tri- iodo-N,N'-bis(2,3-dihydroxypropyl)isophthalamide and Metrizamide ® . 12. The composition of claim 8 wherein the physio¬ logical cold storage solution is selected from the group UW solution and Eurocollins. 13. The composition of claim 8 wherein said first, or lower solution comprises 5-(N-2,3-dihydroxypropylacet- amido) -2,4,5-tri-iodo-N,N'-bis(2, 3-dihydroxypropyl) - isophthalamide in solution with UW solution. 14. The composition of claim 8 wherein the first and second solutions are slightly hypertonic to both cell types. 15. An aqueous composition, initially present as a two-phase gradient, useful in the isolation and purifica¬ tion of islet cells from acinar cells wherein the acinar cells have achieved a higher density than the islet cells through incubation in a first, or lower solution, said composition comprising: A) islet and acinar cells added to said first, or lower solution having a density between 1.097 and 1.126 g/ml, and being slightly hypertonic to the islet and acinar cells, said first solution com¬ prising a mixture of a water soluble, metabolically inert substance selected from 5-(N-2,3-dihydroxy¬ propylacetamido) 2,4,5-tri-iodo-N,N'-bis(2, 3- dihydroxypropyl)isophthalamide and Metrizamide ® , mixed with a physiological cold storage solution selected from UW solution and Eurocollins; B) and a second, or upper solution, having a density of 1.008 g/ml or less, selected from the group comprising: 1) a physiological solution capable of main¬ taining viable cells selected from the group consisting of Hank's solution and tissue culture media, and 2) a mixture of a water soluble, metabolically inert substance selected from 5-(N-2,3-dihy- droxypropylacetamido) 2,4,5-tri-iodo-N,N'- bis(2,3-dihydroxypropyl)isophthalamide and Metrizamide, mixed with a physiological cold storage solution selected from UW solution and Eurocollins, overlayed with a physiological solution capable of maintaining viable cells, selected from the group consisting of Hank's solution and tissue culture media. 16. A method of isolating and purifying one first desired type of cells from a population containing a second type of cells having a different density from said first type after incubation in a first or lower solution, said method comprising: A) adding said population of cells to a first or lower solution comprising a water soluble, metabolic¬ ally inert substance which maintains density, osmo¬ larity and pH in the presence of cells, dissolved in a physiological solution comprising viscosity modi¬ fiers to increase the viscosity of said first solu- tion and osmotically active impermeant agents to suppress cold-induced cellular edema, said first solution having a density greater than that of both cell types in their natural state, but less than that of the second cell type after incubation in said solution, and being slightly hypertonic to both cell types; B) layering a second solution, having a density less than that of both cell types, and being slightly hypertonic to both cell types, selected from the group comprising: 1) a physiological solution capable of maintaining viable cells, and 2) a water soluble, metabolically inert substance which maintains density, osmolarity and pH in the presence of cells, dissolved in a physiological solution comprising viscosity modifiers to increase the viscosity of said second solution and osmotically active imper¬ meant agents to suppress cold-induced cellular edema, overlayed with a physiological solution capable of maintaining viable cells; C) separating the two cell types by applying at least lg force to said combination of solutions for sufficient time to separate said two types of cells whereby said first, desired type of cells collect at or near the interface between said first solution and said second solution and said second type of cells accumulate at or near the bottom below said first solution; and D) removing said first, desired type of cells. 17. The method of claim 16 wherein said second type of cells achieves a higher density than said first type of cells by being made more permeable. 18. The method of claim 16 wherein the first and second solutions are slightly hypertonic to both cell types. 19. The method of claim 18 wherein the cells are made more permeable by preferential digestion with enzymes. 20. The method of claim 16 wherein said physio- logical solution is selected from Hank's solution and tissue culture media. 21. The method of claim 16 wherein the water soluble, metabolically inert substance is selected from the group 5-(N-2,3-dihydroxypropylacetamido) 2,4,5-tri- iodo-N,N'-bis(2,3-dihydroxypropyl) isophthalamide and Metrizamide ® . 22. The method of claim 16 wherein the physiological cold storage solution is selected from the group UW solution and Eurocollins. 23. A method of isolating islet cells from a popu¬ lation containing acinar cells, wherein the acinar cells are made more permeable than the islet cells, said method comprising: A) adding said population of cells to a first or lower solution comprising a water soluble, metabol¬ ically inert substance which maintains density, osmolarity and pH in the presence of cells, dissolved in a physiological solution comprising viscosity modifiers to increase the viscosity of said first solution and osmotically active impermeant agents to suppress cold-induced cellular edema, said first solution having a density in the range of 1.097 to 1.126 g/ml, and being slightly hypertonic to the islet and acinar cells; B) layering a second solution, having a density of 1.008 g/ml or less, and being slightly hypertonic to the islet and acinar cells, selected from the group comprising: 1) a physiological solution capable of maintaining viable cells, and 2) a water soluble, metabolically inert substance which maintains density, osmolarity and pH in the presence of cells, dissolved in a physiological solution comprising viscosity modifiers to increase the viscosity of said second solution and osmotically active imper¬ meant agents to suppress cold-induced cellular edema, overlayed with a physiological solution capable of maintaining viable cells; C) separating the two cell types by applying at least lg force to said combination of solutions for sufficient time to separate said two types of cells whereby said islet cells collect at or near the interface between said first solution and said second solution and said acinar cells accumulate at or near the bottom below said first solution; and D) removing said islet cells. 24. The method of claim 23 wherein said physio¬ logical solution is selected from Hank's solution and tissue culture media. 25. The method of claim 23 wherein said force is between 1 g for 30 minutes and 180 g for 15 minutes at the interface. 26. The method of claim 23 wherein the water soluble, metabolically inert substance is selected from the group 5-(N-2,3-dihydroxypropylacetamido) 2,4,5-tri- iodo-N,N'-bis(2,3-dihydroxypropyl) isophthalamide and Metrizamide ® . 27. The method of claim 23 wherein the physiological cold storage solution is selected from the group UW solution and Eurocollins. 28. The method of claim 23 wherein the viscosity modifiers are colloids. 29. The method of claim 28 wherein the colloids are selected from the group hydroxyethyl starch and serum proteins. 30. The method of claim 23 wherein the viscosity modifiers are polymers. 31. The method of claim 30 wherein the polymers are selected from the group polyethylene glycol, dextran and alginate. 32. The method of claim 23 wherein the osmotically active impermeants are sugars. 33. The method of claim 32 wherein the sugars are selected from the group glucose, mannitol, and sucrose. 34. The method of claim 23 wherein the osmotically active impermeants are anions. 35. The method of claim 34 wherein the anions are selected from the group phosphate, sulfate, glycerophos- phate, citrate and gluconate. 36. The method of claim 23 wherein the osmotically active impermeants are compounds which supress cold induced cellular edema. 37. The method of claim 36 wherein the compounds are selected from lactobionate acid and raffinose. 38. The method of claim 23 wherein said first solution comprises 5-(N-2,3-dihydroxypropylacetamido)- 2,4,5-tri-iodo-N,N'-bis(2,3-dihydroxypropyl)isophthalamide in solution with UW solution. 39. The method of claim 23 wherein the acinar cells are made more permeable by directing collagenase enzymes to acinar cells via ductal injection of said enzymes into a pancreas. 40. The method of claim 23 wherein the first and second solutions are slightly hypertonic to both cell types. 41. A method of isolating islet cells from a population containing acinar cells, wherein the acinar cells have been made more permeable by ductal injection of collagenase into a pancreas, said method comprising: A) adding said population of cells to a first or lower solution, having a density in the range of 1.097 to 1.126 g/ml, and being slightly hypertonic to the islet and acinar cells, comprising a mixture of a water soluble, metabolically inert substance selected from 5-(N-2,3-dihydroxypropylacetamido) 2,4, 5-tri-iodo-N,N' -bis (2 , 3-dihydroxypropyl) - isophthalamide and Metrizamide ® , mixed with a physiological cold storage solution selected from UW solution and Eurocollins; B) layering a second solution, having a density of 1.008 g/ml or less, and being slightly hypertonic to the islet and acinar cells, selected from the group comprising: 1) a physiological solution capable of maintaining viable cells selected from the group consisting of Hank's solution and tissue culture media, and 2) a mixture of a water soluble, metabolically inert substance selected from 5-(N-2,3- dihydroxypropylacetamido) 2,4,5-tri-iodo-N,N'- bis( ,3-dihydroxypropyl) isophthalamide and Metrizamide ® , mixed with a physiological cold storage solution selected from UW solution and Eurocollins, overlayed with a physiological solution capable of maintaining viable cells selected from the group consisting of Hank's solution and tissue culture media; C) separating the two cell types by applying a force, between lg for at least 30 minutes and 18Og for at least 15 minutes, to said combination of solutions whereby said islet cells collect at or near the interface between said first solution and said second solution and said acinar cells accumulate at or near the bottom below said first solution; and D) removing said islet cells.

Inventors: SOON-SHIONG PATRIC, SOON-SHIONG, PATRIC
Applicants: TRANCEL CORP, TRANCEL CORPORATION
Application year: 1990
Main classification: C12N5
IPC codes: A01N1, A61K35, C12N5, C12Q1