WO1996034093A1 - Composition containing collagenase and chymopapain for isolating hepatocytes and pancreatic islet cells - Google Patents

Composition containing collagenase and chymopapain for isolating hepatocytes and pancreatic islet cells Download PDF

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Publication number
WO1996034093A1
WO1996034093A1 PCT/US1996/005725 US9605725W WO9634093A1 WO 1996034093 A1 WO1996034093 A1 WO 1996034093A1 US 9605725 W US9605725 W US 9605725W WO 9634093 A1 WO9634093 A1 WO 9634093A1
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Prior art keywords
nkat
collagenase
chymopapain
tissue
activity
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PCT/US1996/005725
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French (fr)
Inventor
Catherine Lee
Cynthia Zerfass
Tan Thanh Dinh
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Baxter International Inc.
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Application filed by Baxter International Inc. filed Critical Baxter International Inc.
Priority to EP96923189A priority Critical patent/EP0767833A1/en
Priority to JP8532679A priority patent/JPH10508205A/en
Publication of WO1996034093A1 publication Critical patent/WO1996034093A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0676—Pancreatic cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/067—Hepatocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes

Definitions

  • the present invention generally relates to proteolytic enzyme compositions and procedures for digesting connec ⁇ tive tissue. More particularly, the present invention is directed to proteolytic enzyme compositions which can be used for reliably and reproducibly isolating hepatocytes and pancreatic islet cells and to the processes used for such cell isolation.
  • Proteolytic enzymes have found wide utility in a vari ⁇ ety of laboratory an clinical applications. Typically these applications involve cell dissociation and related therapeutic procedures which are benefitted by the ability of proteolytic enzymes to hydrolytically break-up or loosen connective tissue networks.
  • bacterial collagenase derived from Clostridium histolyticum has been used to disperse cells in laboratory tissue culture appli ⁇ cations.
  • collagenase has demonstrated util ⁇ ity in cell isolation procedures such as those associated with isolating pancreatic islets and dispersing a variety of tumor cells.
  • Other uses for collagenase involve its topical use in clinical applications in which collagenase compositions are applied in the treatment of burns or ulcers and wound healing.
  • chymopapain the major proteolytic component of the crude latex of Carica papaya, has been utilized in the treatment of abnormal or herniated discs to selectively dissolve the nucleus pulposus of the disc.
  • Other uses associated with chymopapain include its utility in cryosurgical healing processes.
  • Combinations of proteolytic enzymes such as composi ⁇ tions of bacterial collagenase and hyaluronidase are reportedly particularly useful for digesting or dissolving prostatic tissue in the treatment of benign prostatic hypertrophy.
  • the combination of these two proteolytic enzymes apparently dissolves prostatic tissue in order to relieve the obstructive symptoms of prostatic hypertrophy.
  • bacterial collagenase derived from Clostri- dium histolyticum has found utility in procedures involv ⁇ ing the dissociation and isolation of microvessel cells embedded in fatty tissues. These procedures generally in ⁇ volve combining fatty tissues having embedded microves- sels, such as liposuctioned fat, with collagenase under conditions which cause the collagenase to disrupt and di ⁇ gest the connective tissue. By carefully separating the cells from the digested tissue, viable microvessel cells are recovered.
  • microvessel cells have found particular utility as a coating on the interior of syn ⁇ thetic small diameter vascular grafts implanted in humans and animals to replace blood vessels.
  • micro- vessel cells are useful as deposits on the surface of bio- medical implant devices in general where they provide an improved biocompatibility to the implant.
  • the microvessel cells contribute to the prevention of protein deposits and related cellular deposits on the implants which are known to occur when foreign materials are placed in contact with blood and tissue. In the case of vascular grafts these deposits can quickly cause the vessel to occlude, resulting in the functional failure of the graft.
  • connective tissues are formed largely of collagen, for which collagenase is specific in its hydro- lytic activity, significant amounts of other proteins and glycoproteins are additionally found in connective tissue matrices. Thus, collagenase alone may not effectively hydrolyze all of the tissue mixtures.
  • collagenase derived from native bacteria dif ⁇ fers widely in its collagen specific hydrolytic activity and the amount and character of impurities, including other proteases and toxins.
  • the protease impurities in crude collagenase contribute to the hydrolysis of minor proteins in connective tissue and actually aid in the di ⁇ gestive process.
  • protease impuri ⁇ ties are active with proteins generally and can damage cells.
  • Proteases can also react with collagenase, causing the crude collagenase to be subject to catalytic degrada ⁇ tion.
  • the toxin impurities associated with crude colla ⁇ genase can be a serious problem for procedures involving both in vivo and in vitro applications. Toxins can dis ⁇ rupt cell membranes, destroy cell viability and generally lower cell yield. Additionally, impurities can contain variable amounts of bacterial DNA, which may cause cell -4 - PATENT
  • Another object of the present invention is to provide proteolytic enzyme compositions capable of digesting con ⁇ nective tissue in a reproducible and predictable manner.
  • the present invention accomplishes the above objec ⁇ tives by providing proteolytic enzyme compositions capable of predictably and reproducibly digesting tissue contain- ing hepatocytes and pancreatic islet cells. Further, the present invention provides processes for digesting tissue and dissociating cells from the connective tissue, provid ⁇ ing efficaciously viable hepatocytes and pancreatic islet cells in high yield. The viable cells thereby provided have utility in a variety of in vivo and in vitro applications.
  • the present invention is based upon the discovery that, although neither collagenase nor chymopapain alone is effective to digest tissue, a mixture of collagenase and chymopapain can be used to safely, reproducibly and reliably digest connective tissue formed of a variety of proteins and glycoprotein extracellular matrix material.
  • compositions of purified collagenase and chymopapain have been found to effectively digest connective tissue and reproducibly dissociate and isolate cells embedded in the tissue, -6 -
  • the isolated cells in high yield. Moreover, because the isolated cells have been processed with compositions of purified enzymes, the cell suspensions are essentially free of the harmful effects of toxins and unknown unreactive materials, making them highly viable and safe for in vivo use.
  • the present invention provides novel enzyme compositions and associated methodologies useful for hydrolyzing connective tissue in biological systems.
  • the enzyme compositions of the present invention principally comprise a combination of collagenase, in an amount sufficient to hydrolyze collagen in the biological system, and chymopapain in an amount sufficient to hydrolyze chymopapain active tissue in the biological system.
  • the collagenase and chymopapain are purified and essentially free of toxic components, such as bacterial DNA and sensitizing antigens, and the collagenase is essentially free of non-collagen specific components. It is also within the scope of the present invention to provide associated processes utilizing these enzyme compositions of collagenase and chymoparain.
  • An exemplary process of the present invention includes enzymatically digesting connective tissue by providing an enzyme compo ⁇ sition of collagenase, essentially free of collagen inac ⁇ tive components and toxins and in an amount sufficient to hydrolyze the collagen present in the connective tissue, and chymopapain essentially free of toxins and in an amount sufficient to hydrolyze chymopapain active tissue in the connective tissue.
  • an enzyme compo ⁇ sition of collagenase essentially free of collagen inac ⁇ tive components and toxins and in an amount sufficient to hydrolyze the collagen present in the connective tissue
  • chymopapain essentially free of toxins and in an amount sufficient to hydrolyze chymopapain active tissue in the connective tissue.
  • a preferred process of the present invention utilizes the above steps to hydrolyze connective tissue in order to dissociate and isolate hepatocytes and pancreatic islet cells embedded in the tissue.
  • hepatocytes and pancreatic islet cells which form part of the mixture are dissociated from the connective tissue and isolated in higher yield and have improved via ⁇ bility when compared with cells isolated from tissue hy- drolyzed according to prior art procedures which utilize crude collagenase.
  • the higher yield of such cells pro ⁇ vided by the processes of the present invention is charac- terized by the increased number of viable healthy cells.
  • the increased yield as well as increased viability and integrity of cells isolated according to the processes of the present invention are readily demonstrated by labora ⁇ tory testing techniques.
  • cell-counting techniques provide cell size information and information relating to the distribution of cell sizes in a given batch of isolated cells.
  • cell function and ef ⁇ ficacy are demonstrated by their biological function, such as production of insulin by pancreatic islet cells in re- sponse to glucose concentration change in culture media. This response is characterized by the ratio of insulin production in the presence of glucose to the base-line value.
  • Fig. 1 illustrates the particle size distribution of rat hepatocyte cells isolated using crude collagenase in accordance with the prior art
  • Fig. 2 illustrates the particle size distribution of rat hepatocyte cells isolated using purified collagenase- chymopapain enzyme compositions in accordance with the present invention
  • Fig. 3 illustrates the static incubation glucose response by porcine pancreatic islet cells isolated using crude collagenase according to the prior art
  • Fig. 4 illustrates the static incubation glucose response by porcine pancreatic islet cells isolated using purified collagenase-chymopapain enzyme compositions according to the present invention.
  • the present invention provides proteolytic enzyme compositions and processes capable of predictably and reproducibly digesting physiological connective tissue in a variety of therapeutic and laboratory applications. These applications range from in vivo therapeutic treat ⁇ ment procedures to techniques which involve dissociating and isolating cells embedded in connective tissue for subsequent laboratory or clinical applications.
  • the compositions and processes of the present inven ⁇ tion are suitable for reproducibly hydrolyzing or digest ⁇ ing a wide variety of collagens, non-collagenous connective tissue proteins, glycoproteins, and extracellular matrix materials.
  • Those skilled in the art will appreciate that the ability to hydrolyze a wide range of proteins and protein mixtures makes the teachings of the present invention widely applicable in a number of in vivo as well as in vitro tissue digestion procedures.
  • compositions and processes of the present inven- tion find particular application in cell dissociation pro ⁇ cedures including laboratory cell culture methods and re- lated cell isolation techniques.
  • cells can be effectively and reproduc ⁇ ibly isolated from a host of different proteinaceous con ⁇ nective tissues and harvested in higher yield with im- proved preservation of the cell membranes.
  • these cells have better viability and are free of toxins and contaminants, when compared with cells isolated using prior art processes.
  • compositions and processes of the present invention are particularly suitable for isolating hepatocytes and pancreatic islet cells embedded in connective tissues for subsequent util ⁇ ity in various clinical procedures such as transplanting hepatocytes or pancreatic islet cells into individuals suffering from liver, pancreatic, or other types of diseases.
  • the enzyme compositions of the present invention include purified collagenase in an amount sufficient to hydrolyze collagen present in the system, and chymopapain in an amount sufficient to hydro- lyze chymopapain active tissue in the system.
  • the colla ⁇ genase and the chymopapain are purified and essentially free of toxic components such as bacterial components and sensitizing antigens.
  • the collagenase is free of collagen inactive components.
  • Preferred exemplary embodiments of the present inven ⁇ tion are solutions of collagenase and chymopapain in a physiologically compatible liquid.
  • Suitable physiologi ⁇ cally compatible liquids include phosphate buffered saline solutions and similar buffered electrolyte solutions hav- ing osmolalities which are compatible with physiological tissue.
  • a particularly suitable commercially available electrolyte solution is Plasmalyte® electrolyte solution available from Baxter-Hyland, having a buffered pH of 7.4 and an osmolarity of 294 mOsmol/L obtained with controlled concentrations of sodium, potassium, magnesium, chloride, acetate, and gluconate ions.
  • addi- tives such as human serum albumin and serum are preferred in many applications.
  • compositions of the present invention include a solution of from about 25 nkat/ml to about 120 nkat/ml purified collagenase and from about 0.15 nkat/ml to about 0.55 nkat/ml purified chymopapain in a suitable pH buf ⁇ fered physiologically compatible liquid.
  • the nkat/ml unit is defined as nanomoles of substrate hydrolyzed per second by 1 ml of enzyme solution under the assay condition used.
  • enzyme activity assays were conducted at 37°C.
  • the enzyme composition is a solution of 30 nkat/ml purified collagenase and 0.3 nkat/ml chymopapain in a solution of about 0.4 wt% human serum albumin in Plasmalyte® electrolyte solution for the isolation of hepatocytes and a solution of from about 25 nkat/ml to about 120 nkat/ml of purified collagenase and about 0.15 nkat/ml to about 0.55 nkat/ml chymopapain for pancreatic islet cells.
  • collagenase is derived from the bac ⁇ terium Clostridium histolyticum and in its crude form dif ⁇ fers from batch to batch in hydrolytic activity and pur ⁇ ity. Uncontrolled amounts of impurities found in crude collagenase may include contaminating bacterial compo ⁇ nents, pigment, pyrogens, proteases, and peptidases, including clostripain, trypsin, and caseinase.
  • purified collagenase suitable for use in the compositions of the present invention, is substantially free of pigment, bacterial components, and nonspecific enzyme activity. Crude collagenase is readily available from a number of commercial sources including Sigma Chemical Company of St.
  • purified collagenase suitable for use in accordance with the present invention exhibits a batch-to-batch uniformity in specificity for collagen as well as toxin-free characteristics.
  • chymopapain a proteolytic enzyme extracted from papaya latex
  • Chymopapain is commercially available in a dry lyophilized state from a number of sources including Sigma Chemical of St. Louis, Missouri.
  • Chymopapain is available in crude, partially purified, and more highly purified forms which differ in the amount of papain, lysozyme pep- tidase A and sensitizing antigens found in the prepara- tion.
  • Chymopapain suitable for use in the present inven ⁇ tion is characterized as having essentially no immunogen ⁇ icity and essentially no toxicity as a result of purifica ⁇ tion processes.
  • Chymopapain from most commercial sources which has been purified using known chromatographic purif- ication processes, provides chymopapain suitable in the practice of the present invention.
  • puri- fied chymopapain can be prepared using, for example, the process described in U.S. Patent No. 4,719,108.
  • connective tissue which holds cells together, is a com ⁇ plex mixture of collagen, other extracellular proteins, glycoproteins, and mucopolysaccharides. Purified colla ⁇ genase alone will not effectively hydrolyze all of this extracellular matrix material. However, it has been dis- covered by the present inventors that by combining puri ⁇ fied collagenase with toxin-free chymopapain a wide range of connective tissue systems and biologically derived raw materials can be predictably digested.
  • the hy ⁇ drolyzed tissues and cells isolated during these hydroly- sis processes are free of antigenic components which can cause anaphylactic shock if present in cells or tissues implanted or digested in vivo. Accordingly, cells iso ⁇ lated in accordance with the present invention for subse ⁇ quent implantation do not present toxic health hazards to their recipients. Similarly, the compositions of the present invention can be utilized for in vivo procedures with little risk of anaphylactic shock.
  • the enzyme compositions of the present invention can be prepared according to processes known in the art. Typically, these processes involve mixing the two enzymes in a selected physiologically compatible liquid such as phosphate buffered normal saline solution or Plasmalyte® electrolyte solution containing human serum albumin (HSA) and CaCl 2 . Then lyophilizing the resulting aqueous solu- tion provides a stable dry enzyme preparation which can be reconstituted with deionized water. Preferably, the com ⁇ positions are reconstituted just prior to their use in order to minimize any degradation that may occur once the enzymes are placed in solution.
  • a selected physiologically compatible liquid such as phosphate buffered normal saline solution or Plasmalyte® electrolyte solution containing human serum albumin (HSA) and CaCl 2 .
  • HSA human serum albumin
  • CaCl 2 CaCl 2
  • the prepared enzyme compositions are main ⁇ tained at reduced temperatures in the range of about 4°C ⁇ 2°C until their use.
  • separate stock solutions of each enzyme which typically include concen ⁇ trated forms of the enzyme in a buffered saline solution, can be prepared in advance and stored frozen at about -80°C. Just prior to use, the solutions are thawed, diluted with a suitable physiologically compatible diluent to a desired enzyme activity or concentration, and then combined, to form the enzyme composition.
  • Suitable diluents include aqueous based solutions buffered to a pH of about 7.4 and having a physiologically compatible osmolarity.
  • the processes of the present invention broadly include providing a composition of the present invention and causing the composition to contact selected tissue for a length of time and at a temperature sufficient to substantially hydrolyze the tissue and permit isolation of hepatocytes and pancreatic islet cells.
  • Preferred exemplary processes in accordance with the teachings of the present invention include digesting connective tissue for the purpose of dissociating and isolating cells embedded in the connective tissue.
  • the compositions of. the present invention provide highly viable cells which are particularly useful for gene therapy and transplanting into humans or animals for therapeutic purposes.
  • pancreatic cells can be isolated from donor pancreases and transplanted into humans or animals for purposes of treating pancreatic related diseases.
  • hepatocytes can be isolated from liver in accordance with known procedures utilizing compositions of the present invention.
  • a most preferred process of the present invention in ⁇ cludes providing an appropriate enzyme composition of the present invention and contacting the enzyme composition with connective tissue for a length of time and at a tem ⁇ perature sufficient to substantially hydrolyze the connec ⁇ tive tissue and to isolate hepatocytes and pancreatic islet cells embedded in the tissue.
  • an exemplary preferred pro ⁇ cess for digesting tissue includes the steps of providing an enzyme composition of a Plasmalyte® electrolyte solu ⁇ tion of about 25 nkat/ml to about 120 nkat/ml purified collagenase, about 0.15 nkat/ml to about 0.55 nkat/ml chy- mopapain and about 0.4 wt% human serum albumin.
  • the enzyme composition at about 37°C is perfused into the organ containing the tissue to be digested, for example, a liver or pancreas, by means of cannulation in an amount of about 1-10 ml of enzyme composition per gram of organ weight until the organ is sufficiently expanded to permit dissection, incubation of the combination of cells and partially hydrolyzed tissue, and isolation of the desired viable hepatocytes or islet cells.
  • Further separation of viable cells from the incubated combination can be accomplished by shaking the incubated combination until hydrolyzed tissue separates from a supernatant containing cells. The cells are then separ ⁇ ated from the supernatants by centrifugation and pipeting off the supernatants.
  • Preferred exemplary processes fur- ther include rinsing the cells with a physiologically compatible liquid prior to their evaluation and use.
  • Tissues which are subject to digestion in accordance with the present invention should have good contact with the enzyme solution. Accordingly, large pieces of tissue should be minced with a pair of scissors prior to further treatment. Additionally, the tissue is preferably rinsed with a physiologically compatible rinsing solution in order to remove visible blood contaminants including clotted blood. Suitable rinsing solutions include those having pH ranges and osmolarity ranges which are compat ⁇ ible with cellular material, such as phosphate buffered saline and Plasmalyte® electrolyte solution.
  • a preferred method for rinsing the mixture involves .transferring the tissue into a sieve-tissue grinder cup and adding phos ⁇ phate buffered saline solution to the mixture while stir ⁇ ring. Excess liquids and blood contaminants are removed by the rinsing and sieving process. The homogenized and rinsed mixture is then prepared for the above-described digestion and cell isolation procedures.
  • hepatocytes and pancre ⁇ atic islet cells isolated from hydrolyzed tissues in accordance with the teachings of the present invention are isolated in higher yields and have greater viability than cells isolated by prior art processes.
  • the enzyme compositions used in the processes of the pres ⁇ ent invention are free of toxins, in the event that iso- lated cells are implanted for therapeutic purposes or are subjected to other in vivo uses, any residual cotrans- planted enzyme composition will not pose the threat of an anaphylactic or other adverse response.
  • the superior physical and functional characteristics of the cells isolated according to the process of the present invention are sometimes demonstrated by the higher yield of cells having expected characteristics as deter ⁇ mined by known cell-counting methods.
  • Other indicators of the superior results obtained by the present invention in- elude the higher insulin production response to glucose concentration in culture media by pancreatic islet cells isolated in accordance with this invention.
  • a higher yield of these cells, as produced by the present invention is an indicator of a highly safe and efficacious process.
  • digesting porcine pancreatic tissue utilizing purified collagenase without chymopapain being present resulted in no porcine islet cells being isolated.
  • a comparable procedure utilizing an enzyme composition of purified collagenase at 100 nkat/ml and chymopapain at 0.3 nkat/ml in an electrolyte solution provided an average of 228,800 islet cells.
  • the resultant superior physical and functional charac ⁇ teristics of cells isolated according to the present in ⁇ vention make them particularly useful for transplanting.
  • the high viability and functional ability of these cells provide a transplant that is less susceptible to func ⁇ tional failure.
  • Sprague Dawley of Fisher 344 male rats 8-16 weeks old, 125-350 G, were brought to an animal facility one week before being used as liver donors and were maintained on a 12-hour dark/light cycle.
  • each rat was anesthetized following a standard animal operating procedure. Heparin, 500 u, was administered by intraperi- toneal injection. The abdominal area was cleaned with betadine and opened through a idline incision. A 16- gauge catheter was inserted into the inferior vena cava and secured with a silk tie. While getting the animal ready, calcium-free HEPES-saline buffer and enzyme solu- tion were warmed in a 37°C water bath.
  • a tubing linked to a pump for perfusion was inserted into buffer or enzyme solution.
  • the other end of the perfusate tubing was con ⁇ nected to a cannula.
  • the flow of calcium-free HEPES- saline buffer, pH 7.4 was initiated at 25 ml/minute for 5- 6 minutes.
  • An incision was made on the diaphragm and the inferior vena cava was clamped in the thorax.
  • the portal vein was cut to allow efflux of the buffer.
  • the perfusion was switched, via stopcock, to the collagenase-chymopapain solution and continued for 5-10 minutes.
  • the liver the average weight of which was about 20 g, was significantly distended and the internal structure began to loosen up and showed signs of collapsing.
  • the liver was carefully dissected free and placed on ice in a ster ⁇ ile petri dish containing William's E. media.
  • the liver capsule was peeled back and the hepatocytes were dispersed by gentle shaking.
  • the liver cell suspension was filtered through a 300 ⁇ m mesh filter into a 50 ml sterile centri ⁇ fuge tube and allowed to settle by gravity for 15 minutes, followed by brief centrifugation.
  • the cells were washed three times with sterile HEPES-saline, and the volume was reduced by 50% after each wash.
  • a small aliquot of cell suspension was mixed into isotonic solution.
  • the cell concentration and size distribution were analyzed with a Coulter Counter fitted with a multisizer unit.
  • a total of 8xi0 7 hepatocytes of 90% viability were isolated using crude collagenase compared to a total of 12xl0 7 hepatocytes at 95% viability using the purified collagenase and chymopapain mixture.
  • Figs. 1 ad 2 illustrate the particle size distribution of hepatocytes isolated using the enzyme composition of the present invention (Fig. 2) compared to using crude collagenase (Fig. 1) .
  • the .viable cells recovered using the collagenase- chymopapain mixture were less damaged and were isolated in a higher yield than those using crude collagenase, as can be seen by comparing the 20-30 ⁇ range of the curve shown in Fig. l, which represents the particle size distribution of hepatocytes isolated using crude collagenase with that of the curve shown in Fig. 2, which represents the parti ⁇ cle size distribution of hepatocytes isolated using the purified collagenase-chymopapain enzyme composition of this invention.
  • the following example illustrates the isolation of porcine pancreatic islet cells.
  • Example 2 Enzyme solutions were prepared according to the proce ⁇ dure of Example 1 except that Hanks balanced salt solution (HBSS) containing CaCl 2 was used instead of HEPES-saline buffer solution.
  • HBSS Hanks balanced salt solution
  • the pancreas was weighed and the duct cannulated.
  • Warm enzyme solution in a volume of 4 ml/g of tissue was used to expand the organ by injecting through the duct using a 30 ml syringe. Run-off solution was collected into a tray.
  • the pancreas was then cut into large pieces approximately one inch in diameter and loaded into a container together with all the enzyme solution used for organ expansion.
  • the container with tissue was carefully sealed and agitated by gentle mechanical shaking in a 37°C water bath for 12 minutes or until the majority of the tissue was digested into small fragments.
  • the digested tissue was filtered through a 3 mm screen into another container, placed on -ice, and digested further by gentle manual stirring.
  • the following example illustrates the effect upon cell function of isolating porcine pancreatic islet cells using the enzyme composition of the present invention compared to using crude collagenase.
  • Example 3 Using the porcine pancreatic cells isolated according to the procedure of Example 2, static incubation glucose responses were plotted over time for cells isolated using crude collagenase, shown in Fig. 3, and for cells isolated using the purified collagenase-chymopapain enzyme composi ⁇ tion of the present invention, shown in Fig. 4.
  • Insulin which is a measure of cell function can be seen to have a higher value in the case of the curve plotted in Fig. 4 than in Fig. 3. This is an indication that the islet cells isolated using the enzyme composition of the present invention function more normally than islet cells isolated using the crude collagenase of the prior art.
  • the following example illustrates the isolation of rat islet cells.
  • Example 2 The procedure of Example 2 was followed except that, due to the small quantities of purified enzyme used, the pancreas was expanded with HBSS solution, then cut into pieces approximately 0.5 cm in diameter. About 2.5 g of tissue was suspended in 10 ml enzyme solution in a vial with screw cap. The vial was carefully sealed and agi ⁇ tated in a 37°C water bath until most of the tissue pieces appeared to be disintegrated. Digestion was also moni ⁇ tored by sampling of tissue suspension periodically and staining the islets with dithizone solution. Incubation and shaking were stopped when the majority of the islets was released but before damage to the islets was detected. The results are shown in Table II.

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Abstract

Proteolytic enzyme compositions and processes for isolating hepatocytes and pancreatic islet cells from tissue containing them are provided. The enzyme compositions include collagenase, which is essentially free of toxins and non-collagen specific components, and chymopapain, which is essentially free of toxins. The enzyme compositions preferably contain an aqueous mixture of collagenase having an activity of about 25 nkat/ml to about 120 nkat/ml, and chymopapain having an activity of about 0.15 nkat/ml to about 0.55 nkat/ml.

Description

COMPOSITION CONTAINING COLLAGENA8E AND CHYMOPAPAIN FOR ISOLATING HEPATOCYTES AND PANCREATIC ISLET CELLS
BACKGROUND OF THE INVENTION This is a continuation-in-part of copending applica¬ tion Serial No. 08/049,015, filed on April 16, 1993.
Field of the Invention The present invention generally relates to proteolytic enzyme compositions and procedures for digesting connec¬ tive tissue. More particularly, the present invention is directed to proteolytic enzyme compositions which can be used for reliably and reproducibly isolating hepatocytes and pancreatic islet cells and to the processes used for such cell isolation.
Description of Relevant Art
Proteolytic enzymes have found wide utility in a vari¬ ety of laboratory an clinical applications. Typically these applications involve cell dissociation and related therapeutic procedures which are benefitted by the ability of proteolytic enzymes to hydrolytically break-up or loosen connective tissue networks. For example, bacterial collagenase derived from Clostridium histolyticum has been used to disperse cells in laboratory tissue culture appli¬ cations. Additionally, collagenase has demonstrated util¬ ity in cell isolation procedures such as those associated with isolating pancreatic islets and dispersing a variety of tumor cells. Other uses for collagenase involve its topical use in clinical applications in which collagenase compositions are applied in the treatment of burns or ulcers and wound healing. Other uses include the treat¬ ment of Peyronie's disease and as an adjunct to cryopros- tatectomy for the removal of retained cryoslough, inter- vertebral discolysis, and in ophthalmic surgery. Like collagenase, chymopapain, the major proteolytic component of the crude latex of Carica papaya, has been utilized in the treatment of abnormal or herniated discs to selectively dissolve the nucleus pulposus of the disc. Other uses associated with chymopapain include its utility in cryosurgical healing processes.
Combinations of proteolytic enzymes such as composi¬ tions of bacterial collagenase and hyaluronidase are reportedly particularly useful for digesting or dissolving prostatic tissue in the treatment of benign prostatic hypertrophy. The combination of these two proteolytic enzymes apparently dissolves prostatic tissue in order to relieve the obstructive symptoms of prostatic hypertrophy.
Recently, bacterial collagenase derived from Clostri- dium histolyticum has found utility in procedures involv¬ ing the dissociation and isolation of microvessel cells embedded in fatty tissues. These procedures generally in¬ volve combining fatty tissues having embedded microves- sels, such as liposuctioned fat, with collagenase under conditions which cause the collagenase to disrupt and di¬ gest the connective tissue. By carefully separating the cells from the digested tissue, viable microvessel cells are recovered.
These viable and intact microvessel cells have found particular utility as a coating on the interior of syn¬ thetic small diameter vascular grafts implanted in humans and animals to replace blood vessels. Similarly, micro- vessel cells are useful as deposits on the surface of bio- medical implant devices in general where they provide an improved biocompatibility to the implant. Apparently the microvessel cells contribute to the prevention of protein deposits and related cellular deposits on the implants which are known to occur when foreign materials are placed in contact with blood and tissue. In the case of vascular grafts these deposits can quickly cause the vessel to occlude, resulting in the functional failure of the graft. One problem associated with the use of commercial sources of crude collagenase to digest fatty tissues, as well as connective tissue in general, is that the degree to which tissue digests or hydrolyzes is unpredictable. Moreover, cells which are isolated from tissue digestion procedures utilizing crude collagenase can be inferior in quality and have a low degree of viability and efficacy. Even when viable cells are successfully isolated, the yield and degree of viability is unpredictable. The unpredictable nature of these procedures may be attributed to the lot variations inherent in commercial sources of crude collagenase. Another factor which may contribute to the lack of reproducibility in these proce¬ dures is the nature of the mixture of tissues being di- gested. While connective tissues are formed largely of collagen, for which collagenase is specific in its hydro- lytic activity, significant amounts of other proteins and glycoproteins are additionally found in connective tissue matrices. Thus, collagenase alone may not effectively hydrolyze all of the tissue mixtures.
Further, collagenase derived from native bacteria dif¬ fers widely in its collagen specific hydrolytic activity and the amount and character of impurities, including other proteases and toxins. The protease impurities in crude collagenase contribute to the hydrolysis of minor proteins in connective tissue and actually aid in the di¬ gestive process. However, unfortunately protease impuri¬ ties are active with proteins generally and can damage cells. Proteases can also react with collagenase, causing the crude collagenase to be subject to catalytic degrada¬ tion. The toxin impurities associated with crude colla¬ genase can be a serious problem for procedures involving both in vivo and in vitro applications. Toxins can dis¬ rupt cell membranes, destroy cell viability and generally lower cell yield. Additionally, impurities can contain variable amounts of bacterial DNA, which may cause cell -4 - PATENT
damage and possible immunological problems when isolated cells or tissue digestion procedures involve in vivo ap¬ plications. Finally, the non-collagenase impurities found in crude collagenase may act as sensitizing antigens which can cause anaphylactic shock if administered to patients. Thus, in view of the varying and unpredictable nature of crude collagenase compositions which contain a host of proteolytically active and unreactive compounds as well as toxins, the use of crude collagenase compositions for therapeutic digestion procedures and cell dissociation techniques can be unreliable. Alternatively, using pur¬ ified collagenase having essentially only collagen speci¬ fic hydrolytic reactive components in these tissue diges¬ tion procedures has not been successful. The failure of purified collagenase in these procedures is apparently due to the tissue containing non-collagen proteins which are not digested by collagen specific collagenase. Crude col¬ lagenase will digest these tissues because it contains other proteolytic enzymes. However, it does so to a varying and unpredictable degree.
It has been suggested that bacteria genetically engineered to produce limited forms of collagenase having known molecular weights and hydrolytic activity may be advantageous when utilized in tissue digestion procedures. However, even when a wide range of isomeric forms of col¬ lagenase are utilized in tissue digestion procedures, the narrow specificity of collagenase in general precludes ef¬ fectively hydrolyzing all of the tissue. This is because the wide spectrum of proteolytic activity and non-collagen specificity supplied by crude collagenase derived from native bacteria is not available in these genetically engineered sources of collagenase. Even when toxins are removed from genetically engineered sources of collagen¬ ase, the resulting collagenase compositions do not provide hydrolytic characteristics suitable for efficacious tissue digestion and/or cell dissociation procedures. Accordingly, it is a principal object of the present invention to provide proteolytic enzyme compositions cap¬ able of dissociating tissue containing viable hepatocytes and pancreatic islet cells with predictable and reprodu- cible yields and quality.
Another object of the present invention is to provide proteolytic enzyme compositions capable of digesting con¬ nective tissue in a reproducible and predictable manner.
It is another object of the present invention to pro- vide tissue digestion procedures and associated therapeu¬ tic procedures which provide reproducible and predictable results.
It is a further object of the present invention to isolate viable and efficacious hepatocytes and pancreatic islet cells for various medical uses.
SUMMARY OF THE INVENTION The present invention accomplishes the above objec¬ tives by providing proteolytic enzyme compositions capable of predictably and reproducibly digesting tissue contain- ing hepatocytes and pancreatic islet cells. Further, the present invention provides processes for digesting tissue and dissociating cells from the connective tissue, provid¬ ing efficaciously viable hepatocytes and pancreatic islet cells in high yield. The viable cells thereby provided have utility in a variety of in vivo and in vitro applications.
The present invention is based upon the discovery that, although neither collagenase nor chymopapain alone is effective to digest tissue, a mixture of collagenase and chymopapain can be used to safely, reproducibly and reliably digest connective tissue formed of a variety of proteins and glycoprotein extracellular matrix material. As a feature of the present invention, compositions of purified collagenase and chymopapain have been found to effectively digest connective tissue and reproducibly dissociate and isolate cells embedded in the tissue, -6 -
providing the isolated cells in high yield. Moreover, because the isolated cells have been processed with compositions of purified enzymes, the cell suspensions are essentially free of the harmful effects of toxins and unknown unreactive materials, making them highly viable and safe for in vivo use.
More particularly, the present invention provides novel enzyme compositions and associated methodologies useful for hydrolyzing connective tissue in biological systems. The enzyme compositions of the present invention principally comprise a combination of collagenase, in an amount sufficient to hydrolyze collagen in the biological system, and chymopapain in an amount sufficient to hydrolyze chymopapain active tissue in the biological system. Preferably, as a feature of the present invention the collagenase and chymopapain are purified and essentially free of toxic components, such as bacterial DNA and sensitizing antigens, and the collagenase is essentially free of non-collagen specific components. It is also within the scope of the present invention to provide associated processes utilizing these enzyme compositions of collagenase and chymoparain. These pro¬ cesses effectively hydrolyze connective tissue systems and dissociate cells embedded in the tissue to produce highly efficacious and viable cells in high yield. An exemplary process of the present invention includes enzymatically digesting connective tissue by providing an enzyme compo¬ sition of collagenase, essentially free of collagen inac¬ tive components and toxins and in an amount sufficient to hydrolyze the collagen present in the connective tissue, and chymopapain essentially free of toxins and in an amount sufficient to hydrolyze chymopapain active tissue in the connective tissue. Contacting the enzyme composi¬ tion with the connective tissue produces a turbid-appear- ing system indicating substantial tissue hydrolysis.
More specifically, a preferred process of the present invention utilizes the above steps to hydrolyze connective tissue in order to dissociate and isolate hepatocytes and pancreatic islet cells embedded in the tissue. Advantage¬ ously, hepatocytes and pancreatic islet cells which form part of the mixture are dissociated from the connective tissue and isolated in higher yield and have improved via¬ bility when compared with cells isolated from tissue hy- drolyzed according to prior art procedures which utilize crude collagenase. The higher yield of such cells pro¬ vided by the processes of the present invention is charac- terized by the increased number of viable healthy cells. The increased yield as well as increased viability and integrity of cells isolated according to the processes of the present invention are readily demonstrated by labora¬ tory testing techniques. More particularly, cell-counting techniques provide cell size information and information relating to the distribution of cell sizes in a given batch of isolated cells. Similarly, cell function and ef¬ ficacy are demonstrated by their biological function, such as production of insulin by pancreatic islet cells in re- sponse to glucose concentration change in culture media. This response is characterized by the ratio of insulin production in the presence of glucose to the base-line value.
The greater viability and number of useful cells iso- lated according to the teachings of the present invention are particularly important for applications which involve various medical procedures such as transplanting hepato¬ cytes or pancreatic islet cells into individuals suffering from liver or pancreatic disease. Further objects, features, and advantages of the present invention will become apparent to those skilled in the art from a consideration of the following detailed description, taken in conjunction with the associated drawings. BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 illustrates the particle size distribution of rat hepatocyte cells isolated using crude collagenase in accordance with the prior art; Fig. 2 illustrates the particle size distribution of rat hepatocyte cells isolated using purified collagenase- chymopapain enzyme compositions in accordance with the present invention;
Fig. 3 illustrates the static incubation glucose response by porcine pancreatic islet cells isolated using crude collagenase according to the prior art; and
Fig. 4 illustrates the static incubation glucose response by porcine pancreatic islet cells isolated using purified collagenase-chymopapain enzyme compositions according to the present invention.
DESCRIPTION OF EXEMPLARY EMBODIMENTS The present invention provides proteolytic enzyme compositions and processes capable of predictably and reproducibly digesting physiological connective tissue in a variety of therapeutic and laboratory applications. These applications range from in vivo therapeutic treat¬ ment procedures to techniques which involve dissociating and isolating cells embedded in connective tissue for subsequent laboratory or clinical applications. The compositions and processes of the present inven¬ tion are suitable for reproducibly hydrolyzing or digest¬ ing a wide variety of collagens, non-collagenous connective tissue proteins, glycoproteins, and extracellular matrix materials. Those skilled in the art will appreciate that the ability to hydrolyze a wide range of proteins and protein mixtures makes the teachings of the present invention widely applicable in a number of in vivo as well as in vitro tissue digestion procedures.
The compositions and processes of the present inven- tion find particular application in cell dissociation pro¬ cedures including laboratory cell culture methods and re- lated cell isolation techniques. As a feature of the present invention, cells can be effectively and reproduc¬ ibly isolated from a host of different proteinaceous con¬ nective tissues and harvested in higher yield with im- proved preservation of the cell membranes. Moreover, these cells have better viability and are free of toxins and contaminants, when compared with cells isolated using prior art processes. For this reason, the compositions and processes of the present invention are particularly suitable for isolating hepatocytes and pancreatic islet cells embedded in connective tissues for subsequent util¬ ity in various clinical procedures such as transplanting hepatocytes or pancreatic islet cells into individuals suffering from liver, pancreatic, or other types of diseases.
More particularly, the enzyme compositions of the present invention include purified collagenase in an amount sufficient to hydrolyze collagen present in the system, and chymopapain in an amount sufficient to hydro- lyze chymopapain active tissue in the system. The colla¬ genase and the chymopapain are purified and essentially free of toxic components such as bacterial components and sensitizing antigens. Additionally, the collagenase is free of collagen inactive components. Preferred exemplary embodiments of the present inven¬ tion are solutions of collagenase and chymopapain in a physiologically compatible liquid. Suitable physiologi¬ cally compatible liquids include phosphate buffered saline solutions and similar buffered electrolyte solutions hav- ing osmolalities which are compatible with physiological tissue. A particularly suitable commercially available electrolyte solution is Plasmalyte® electrolyte solution available from Baxter-Hyland, having a buffered pH of 7.4 and an osmolarity of 294 mOsmol/L obtained with controlled concentrations of sodium, potassium, magnesium, chloride, acetate, and gluconate ions. As illustrated below, addi- tives such as human serum albumin and serum are preferred in many applications.
Those skilled in the art will appreciate that the con¬ centration or amount of each enzyme present in the solu- tions will vary with the amount and the type of tissue to be hydrolyzed. The well-known principles of enzyme activ¬ ity are applicable and basic experimentation involving techniques designed to optimize enzyme concentration and total activity provide necessary information to assure the effective hydrolysis of the amount and type of selected connective tissue. For applications directed toward di¬ gesting connective tissue and isolating hepatocytes and pancreatic islet cells embedded in the tissue, exemplary compositions of the present invention include a solution of from about 25 nkat/ml to about 120 nkat/ml purified collagenase and from about 0.15 nkat/ml to about 0.55 nkat/ml purified chymopapain in a suitable pH buf¬ fered physiologically compatible liquid. The nkat/ml unit is defined as nanomoles of substrate hydrolyzed per second by 1 ml of enzyme solution under the assay condition used. For purposes of the present invention, enzyme activity assays were conducted at 37°C. In a preferred embodiment of the present invention, the enzyme composition is a solution of 30 nkat/ml purified collagenase and 0.3 nkat/ml chymopapain in a solution of about 0.4 wt% human serum albumin in Plasmalyte® electrolyte solution for the isolation of hepatocytes and a solution of from about 25 nkat/ml to about 120 nkat/ml of purified collagenase and about 0.15 nkat/ml to about 0.55 nkat/ml chymopapain for pancreatic islet cells.
Most commercial collagenase is derived from the bac¬ terium Clostridium histolyticum and in its crude form dif¬ fers from batch to batch in hydrolytic activity and pur¬ ity. Uncontrolled amounts of impurities found in crude collagenase may include contaminating bacterial compo¬ nents, pigment, pyrogens, proteases, and peptidases, including clostripain, trypsin, and caseinase. On the other hand purified collagenase, suitable for use in the compositions of the present invention, is substantially free of pigment, bacterial components, and nonspecific enzyme activity. Crude collagenase is readily available from a number of commercial sources including Sigma Chemical Company of St. Louis, Missouri, and Boehringer Mannheim Biochemicals of Indianapolis, Indiana. Commer¬ cial sources of crude forms of collagenase are typically packaged in a lyophilized powder form which helps maintain its stability. Alternative sources of collagenase which are substantially free of toxins and collagen inactive compounds include collagenase purified according to the process described in Bond et al. "Purification and Separa¬ tion of Individual Collagenases of Clostridium histolyti- cum Using Red Dye Ligand Chromatography", Biochemistry Vol. 23, No. 13, 3077-3091, 1984. By removing non¬ specific enzyme activities and minimizing or eliminating sensitizing antigens in order to minimize immunogenicity, purified collagenase suitable for use in accordance with the present invention exhibits a batch-to-batch uniformity in specificity for collagen as well as toxin-free characteristics.
Similarly, chymopapain, a proteolytic enzyme extracted from papaya latex, is commercially available in a dry lyophilized state from a number of sources including Sigma Chemical of St. Louis, Missouri. Chymopapain is available in crude, partially purified, and more highly purified forms which differ in the amount of papain, lysozyme pep- tidase A and sensitizing antigens found in the prepara- tion. Chymopapain suitable for use in the present inven¬ tion is characterized as having essentially no immunogen¬ icity and essentially no toxicity as a result of purifica¬ tion processes. Chymopapain from most commercial sources, which has been purified using known chromatographic purif- ication processes, provides chymopapain suitable in the practice of the present invention. Alternatively, puri- fied chymopapain can be prepared using, for example, the process described in U.S. Patent No. 4,719,108.
An exemplary tissue system demonstrating the features of the present invention is connective tissue. Generally, connective tissue, which holds cells together, is a com¬ plex mixture of collagen, other extracellular proteins, glycoproteins, and mucopolysaccharides. Purified colla¬ genase alone will not effectively hydrolyze all of this extracellular matrix material. However, it has been dis- covered by the present inventors that by combining puri¬ fied collagenase with toxin-free chymopapain a wide range of connective tissue systems and biologically derived raw materials can be predictably digested. Moreover, the hy¬ drolyzed tissues and cells isolated during these hydroly- sis processes are free of antigenic components which can cause anaphylactic shock if present in cells or tissues implanted or digested in vivo. Accordingly, cells iso¬ lated in accordance with the present invention for subse¬ quent implantation do not present toxic health hazards to their recipients. Similarly, the compositions of the present invention can be utilized for in vivo procedures with little risk of anaphylactic shock.
The enzyme compositions of the present invention can be prepared according to processes known in the art. Typically, these processes involve mixing the two enzymes in a selected physiologically compatible liquid such as phosphate buffered normal saline solution or Plasmalyte® electrolyte solution containing human serum albumin (HSA) and CaCl2. Then lyophilizing the resulting aqueous solu- tion provides a stable dry enzyme preparation which can be reconstituted with deionized water. Preferably, the com¬ positions are reconstituted just prior to their use in order to minimize any degradation that may occur once the enzymes are placed in solution. Similarly, it is pre- ferred that the prepared enzyme compositions are main¬ tained at reduced temperatures in the range of about 4°C ± 2°C until their use. Alternatively, separate stock solutions of each enzyme, which typically include concen¬ trated forms of the enzyme in a buffered saline solution, can be prepared in advance and stored frozen at about -80°C. Just prior to use, the solutions are thawed, diluted with a suitable physiologically compatible diluent to a desired enzyme activity or concentration, and then combined, to form the enzyme composition. Suitable diluents include aqueous based solutions buffered to a pH of about 7.4 and having a physiologically compatible osmolarity.
It is additionally within the scope of the present invention to utilize these compositions in processes for digesting connective tissue. Thus, the processes of the present invention broadly include providing a composition of the present invention and causing the composition to contact selected tissue for a length of time and at a temperature sufficient to substantially hydrolyze the tissue and permit isolation of hepatocytes and pancreatic islet cells. Preferred exemplary processes in accordance with the teachings of the present invention include digesting connective tissue for the purpose of dissociating and isolating cells embedded in the connective tissue. When used in accordance with processes known in the art for dissociating and isolating cells, the compositions of. the present invention provide highly viable cells which are particularly useful for gene therapy and transplanting into humans or animals for therapeutic purposes. For example, pancreatic cells can be isolated from donor pancreases and transplanted into humans or animals for purposes of treating pancreatic related diseases. Additionally, hepatocytes can be isolated from liver in accordance with known procedures utilizing compositions of the present invention. A most preferred process of the present invention in¬ cludes providing an appropriate enzyme composition of the present invention and contacting the enzyme composition with connective tissue for a length of time and at a tem¬ perature sufficient to substantially hydrolyze the connec¬ tive tissue and to isolate hepatocytes and pancreatic islet cells embedded in the tissue. For purposes of dis- sociating and isolating cells, an exemplary preferred pro¬ cess for digesting tissue includes the steps of providing an enzyme composition of a Plasmalyte® electrolyte solu¬ tion of about 25 nkat/ml to about 120 nkat/ml purified collagenase, about 0.15 nkat/ml to about 0.55 nkat/ml chy- mopapain and about 0.4 wt% human serum albumin. Then, the enzyme composition at about 37°C is perfused into the organ containing the tissue to be digested, for example, a liver or pancreas, by means of cannulation in an amount of about 1-10 ml of enzyme composition per gram of organ weight until the organ is sufficiently expanded to permit dissection, incubation of the combination of cells and partially hydrolyzed tissue, and isolation of the desired viable hepatocytes or islet cells.
Further separation of viable cells from the incubated combination can be accomplished by shaking the incubated combination until hydrolyzed tissue separates from a supernatant containing cells. The cells are then separ¬ ated from the supernatants by centrifugation and pipeting off the supernatants. Preferred exemplary processes fur- ther include rinsing the cells with a physiologically compatible liquid prior to their evaluation and use.
Tissues which are subject to digestion in accordance with the present invention should have good contact with the enzyme solution. Accordingly, large pieces of tissue should be minced with a pair of scissors prior to further treatment. Additionally, the tissue is preferably rinsed with a physiologically compatible rinsing solution in order to remove visible blood contaminants including clotted blood. Suitable rinsing solutions include those having pH ranges and osmolarity ranges which are compat¬ ible with cellular material, such as phosphate buffered saline and Plasmalyte® electrolyte solution. A preferred method for rinsing the mixture involves .transferring the tissue into a sieve-tissue grinder cup and adding phos¬ phate buffered saline solution to the mixture while stir¬ ring. Excess liquids and blood contaminants are removed by the rinsing and sieving process. The homogenized and rinsed mixture is then prepared for the above-described digestion and cell isolation procedures.
As generally mentioned above, hepatocytes and pancre¬ atic islet cells isolated from hydrolyzed tissues in accordance with the teachings of the present invention are isolated in higher yields and have greater viability than cells isolated by prior art processes. Moreover, since the enzyme compositions used in the processes of the pres¬ ent invention are free of toxins, in the event that iso- lated cells are implanted for therapeutic purposes or are subjected to other in vivo uses, any residual cotrans- planted enzyme composition will not pose the threat of an anaphylactic or other adverse response.
The superior physical and functional characteristics of the cells isolated according to the process of the present invention are sometimes demonstrated by the higher yield of cells having expected characteristics as deter¬ mined by known cell-counting methods. Other indicators of the superior results obtained by the present invention in- elude the higher insulin production response to glucose concentration in culture media by pancreatic islet cells isolated in accordance with this invention. Furthermore, since intact and viable hepatocytes or pancreatic islet cells can be isolated by the process of the present inven- tion, a higher yield of these cells, as produced by the present invention, is an indicator of a highly safe and efficacious process.
As described in more detail in the examples which follow, digesting porcine pancreatic tissue utilizing purified collagenase without chymopapain being present resulted in no porcine islet cells being isolated. In contrast, a comparable procedure utilizing an enzyme composition of purified collagenase at 100 nkat/ml and chymopapain at 0.3 nkat/ml in an electrolyte solution provided an average of 228,800 islet cells. These exem¬ plary results demonstrate that islet cells can be effec- tively isolated according to the process of the present invention.
The resultant superior physical and functional charac¬ teristics of cells isolated according to the present in¬ vention make them particularly useful for transplanting. The high viability and functional ability of these cells provide a transplant that is less susceptible to func¬ tional failure.
The invention will be better understood by reference to the following nonlimiting examples which illustrate the use of exemplary enzyme compositions of the present inven¬ tion in processes for isolating hepatocytes and pancreatic islet cells from tissue containing them. In these exam¬ ples the activity of collagenase is expressed as nkat/ml of enzyme composition as determined by assay using FALGPA, furylacryloyl Leu-Gly-Pro-Ala synthetic substrate. The ac¬ tivity of chymopapain was determined by assay using BAPNA, Benzoyl-L-arginine-p-nitroanilide synthetic substrate.
The following example demonstrates the isolation of hepatocytes according to the present invention.
Example 1
Purified collagenase and chymopapain, preactivated with 5 mM L-cysteine, were diluted into isotonic HEPES- saline buffer, pH 7.4, containing 5 mM CaCl2 to a final concentration of 30 nkat/ml for collagenase and 0.3 nkat/ml for chymopapain. Crude collagenase was weighed and dissolved in the same buffer system to 0.4 mg/ml. The solutions were filtered through 0.2 micron filters for sterilization and kept on ice until use.
Sprague Dawley of Fisher 344 male rats, 8-16 weeks old, 125-350 G, were brought to an animal facility one week before being used as liver donors and were maintained on a 12-hour dark/light cycle. For liver perfusion, each rat was anesthetized following a standard animal operating procedure. Heparin, 500 u, was administered by intraperi- toneal injection. The abdominal area was cleaned with betadine and opened through a idline incision. A 16- gauge catheter was inserted into the inferior vena cava and secured with a silk tie. While getting the animal ready, calcium-free HEPES-saline buffer and enzyme solu- tion were warmed in a 37°C water bath. A tubing linked to a pump for perfusion was inserted into buffer or enzyme solution. The other end of the perfusate tubing was con¬ nected to a cannula. The flow of calcium-free HEPES- saline buffer, pH 7.4 was initiated at 25 ml/minute for 5- 6 minutes. An incision was made on the diaphragm and the inferior vena cava was clamped in the thorax. The portal vein was cut to allow efflux of the buffer. The perfusion was switched, via stopcock, to the collagenase-chymopapain solution and continued for 5-10 minutes. At this time the liver, the average weight of which was about 20 g, was significantly distended and the internal structure began to loosen up and showed signs of collapsing. The liver was carefully dissected free and placed on ice in a ster¬ ile petri dish containing William's E. media. The liver capsule was peeled back and the hepatocytes were dispersed by gentle shaking. The liver cell suspension was filtered through a 300 μm mesh filter into a 50 ml sterile centri¬ fuge tube and allowed to settle by gravity for 15 minutes, followed by brief centrifugation. The cells were washed three times with sterile HEPES-saline, and the volume was reduced by 50% after each wash. A small aliquot of cell suspension was mixed into isotonic solution. The cell concentration and size distribution were analyzed with a Coulter Counter fitted with a multisizer unit. A total of 8xi07 hepatocytes of 90% viability were isolated using crude collagenase compared to a total of 12xl07 hepatocytes at 95% viability using the purified collagenase and chymopapain mixture.
Figs. 1 ad 2 illustrate the particle size distribution of hepatocytes isolated using the enzyme composition of the present invention (Fig. 2) compared to using crude collagenase (Fig. 1) .
The .viable cells recovered using the collagenase- chymopapain mixture were less damaged and were isolated in a higher yield than those using crude collagenase, as can be seen by comparing the 20-30 μ range of the curve shown in Fig. l, which represents the particle size distribution of hepatocytes isolated using crude collagenase with that of the curve shown in Fig. 2, which represents the parti¬ cle size distribution of hepatocytes isolated using the purified collagenase-chymopapain enzyme composition of this invention.
The following example illustrates the isolation of porcine pancreatic islet cells.
Example 2 Enzyme solutions were prepared according to the proce¬ dure of Example 1 except that Hanks balanced salt solution (HBSS) containing CaCl2 was used instead of HEPES-saline buffer solution.
Female farm pigs, six months of age, were brought to an animal facility one week before being used as pancreas donors. The pigs were fasted overnight prior to pancrea- tomy. The tail and body of the pancreas will be removed surgically with no warm-ischemia time. After removal , the pancreas will be placed in cold University of Wisconsin (UW) solution on ice and immediately transported to the laboratory.
After removing the surrounding tissue, the pancreas was weighed and the duct cannulated. Warm enzyme solution in a volume of 4 ml/g of tissue was used to expand the organ by injecting through the duct using a 30 ml syringe. Run-off solution was collected into a tray. The pancreas was then cut into large pieces approximately one inch in diameter and loaded into a container together with all the enzyme solution used for organ expansion. The container with tissue was carefully sealed and agitated by gentle mechanical shaking in a 37°C water bath for 12 minutes or until the majority of the tissue was digested into small fragments. The digested tissue was filtered through a 3 mm screen into another container, placed on -ice, and digested further by gentle manual stirring. During cold digestion, small cell clumps, including free islets, were separated every three minutes by filtering the top half of the digest through an 800 μm screen and stored in cold preservation solution until all tissues were digested. To the remaining digest, an equal amount of cold preservation solution was added and digestion continued until the majority of the islets were free. The islet suspensions obtained were pooled. The appearance of digested tissue and the presence of free islets were monitored microscop¬ ically, using dithizone (DTH) staining to identify β cells. The yield was estimated by the number of stained islets in suspension of known volume. The remaining un¬ digested tissue was weighed to determine the weight of di¬ gested tissue. The results obtained are shown in Table I.
TABLE I.
Figure imgf000021_0001
The variation in tissue digested and total islets released was due largely to the inherited differences among individual animals. In contrast to the data shown in Table I, when crude collagenase was used, it either did not show any effectiveness, or needed to be used at widely varying concentrations for islet isolation. No reprodu¬ cible yields were obtained using crude collagenase.
The following example illustrates the effect upon cell function of isolating porcine pancreatic islet cells using the enzyme composition of the present invention compared to using crude collagenase.
Example 3 Using the porcine pancreatic cells isolated according to the procedure of Example 2, static incubation glucose responses were plotted over time for cells isolated using crude collagenase, shown in Fig. 3, and for cells isolated using the purified collagenase-chymopapain enzyme composi¬ tion of the present invention, shown in Fig. 4. The index for insulin production: Insulin (stimulated)
Insulin (unstimulated) which is a measure of cell function can be seen to have a higher value in the case of the curve plotted in Fig. 4 than in Fig. 3. This is an indication that the islet cells isolated using the enzyme composition of the present invention function more normally than islet cells isolated using the crude collagenase of the prior art.
The following example illustrates the isolation of rat islet cells.
Example 4
The procedure of Example 2 was followed except that, due to the small quantities of purified enzyme used, the pancreas was expanded with HBSS solution, then cut into pieces approximately 0.5 cm in diameter. About 2.5 g of tissue was suspended in 10 ml enzyme solution in a vial with screw cap. The vial was carefully sealed and agi¬ tated in a 37°C water bath until most of the tissue pieces appeared to be disintegrated. Digestion was also moni¬ tored by sampling of tissue suspension periodically and staining the islets with dithizone solution. Incubation and shaking were stopped when the majority of the islets was released but before damage to the islets was detected. The results are shown in Table II.
TABLE II.
Collagenase Chymopapain
Experiment Concentration Concentration No. of
(nkat/ml) (nkat/ml) Islets Viability
A 50 0.25 1089 94%
B 50 0.15 325 99% c 50 0.40 933 91%
Although there has been hereinabove described a compo¬ sition containing collagenase and chymopapain for hydro- lyzing connective tissue to isolate cells in accordance with the present invention, for the purpose of illustrat¬ ing the manner in which the invention may be used to ad- vantage, it should be appreciated that the invention is not limited thereto. Accordingly, any and all modifica¬ tions, variations, or equivalent arrangements which may occur to those skilled in the art, should be considered to be within the scope of the present invention as defined in the appended claims.

Claims

WE CLAIM :
1. An enzyme composition useful for hydrolyzing tissue containing hepatocytes or pancreatic islet cells, said composition comprising: an aqueous mixture of collagenase having an activity of about 25 nkat/ml to about 120 nkat/ml, and chymopapain having an activity of about 0.15 nkat/ml to about 0.55 nkat/ml, in a physio¬ logically compatible electrolyte solution buffered to a pH of about 7.0 to 7.4, wherein the activity of collagenase in nkat/ml is defined as the number of nanomoles of furylacryloyl Leu-Gly-Pro-Ala syn¬ thetic substrate (FALGPA) hydrolyzed per second by 1 ml of collagenase solution at 37°C and the acti¬ vity of chymopapain in nkat/ml is defined as the number of nanomoles of Benzoyl-L-arginine-p-nitro- anilide synthetic substrate (BAPNA) hydrolyzed per second by 1 ml of chymopapain solution at 37°c, said collagenase being essentially free of toxins and non-collagen active components and said chymopapain being essentially free c.f toxins.
2. The enzyme composition of claim 1 wherein said electrolyte solution is a phosphate buffered saline solution.
3. The enzyme composition of claim 1 wherein said electrolyte solution is a HEPES-saline buffer, pH 7.4 containing CaCl2.
4. The enzyme composition of claim 1 wherein said electrolyte solution is a Hanks balanced salt solution containing CaCl2.
5. The enzyme composition of claim l wherein said collagenase has an activity of about 30 nkat/ml and said chymopapain has an activity of about 0.3 nkat/ml.
6. A process for isolating viable hepatocytes or pancreatic islet cells from tissue containing viable hepatocytes or pancreatic islet cells, said process comprising the steps of: providing an enzyme composition comprising an aqueous mixture of collagenase having an activity of about 25 nkat/ml to about 120 nkat/ml, and chymopapain having an activity of about 0.15 nkat/ml to about 0.55 nkat/ml, in a physiologically compatible electro- lyte solution buffered to a pH of about 7.0 to 7.4, said collagenase being essentially free of toxins and non-collagen active components and said chymopapain being essentially free of toxins; contacting tissue containing viable hepato- cytes or pancreatic islet cells with said enzyme composition for a length of time and at a tempera¬ ture sufficient to hydrolyze said tissue without adversely affrcting the viability of said hepato¬ cytes or pancreatic islet cells; and isolating viable hepatocytes or pancreatic islet cells from the hydrolyzed tissue.
7. The process of claim 6 wherein said electrolyte solution is a phosphate buffered saline solution.
8. The process of claim 6 wherein said electrolyte solution is a HEPES-saline buffer, pH 7.4 containing CaCl2.
9. The process of claim 6 wherein said electrolyte solution is a Hanks balanced salt solution containing CaCl2.
10. The process of claim 6 wherein said collagenase has an activity of 30 nkat/ml and said chymopapain has an activity of 0.3 nkat/ml.
11. The process of claim 6 wherein the tissue-con¬ tacting step is performed by means of perfusion of an organ containing said tissue.
PCT/US1996/005725 1995-04-25 1996-04-24 Composition containing collagenase and chymopapain for isolating hepatocytes and pancreatic islet cells WO1996034093A1 (en)

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