JPS6152737B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a reversible encapsulation method, i.e., encapsulating a core material, which may be an individual cell, tissue fragment or microorganism, and later releasing this material by selectively disrupting the capsule membrane. Concerning encapsulation methods that can be used for release.

One important embodiment of the present invention involves microencapsulating viable cells so that they can later be released from within the manufactured capsule membrane without disruption.

U.S. patent application Ser. Discloses microencapsulation techniques that can be used to encapsulate. A significant advantage of this method is that the conditions under which the capsule membrane is formed do not include any toxic or denaturing agents, extreme temperatures or other conditions that would destroy living cells. The method of this application is therefore well suited for microencapsulating living materials that remain viable and in a healthy state. Since this method allows some control over membrane permeability, it is now possible to
Microencapsulation of cell cultures of eukaryotic cells or other sources protects the cells in the culture from contaminating bacteria, high molecular weight immunoglobulins, and other potentially toxic factors, and protects them from continued viability and ongoing development. can be confined within a minimal environment that is sufficiently compatible with the metabolic functions of the human body. When the microcapsules are suspended in a conventional culture medium sufficient to promote the growth of the viable cells encapsulated, the microencapsulated cells take up substances needed for metabolism and diffuse through the membrane; Metabolic products are free to be excreted through the capsule membrane into the surrounding medium.

The present invention relates to specific membranes synthesized during the microencapsulation process described in the above-mentioned applications without appreciable disruption to the core material, which is the encapsulated individual cells, tissue fragments or microorganisms. It relates to a method of selectively destroying. Specifically, the method of the present invention comprises at least two water-soluble components, i.e., a polymer containing a plurality of cationic moieties;
That is, a membrane consisting of a water-insoluble matrix formed from polylysine and a polymer with multiple anionic moieties, namely sodium alginate gum. The two components are joined by salt bridges between the anionic and cationic moieties to form a matrix.

The method of the present invention uses membranes of the type described above for cations,
The method includes exposure to a solution of polyvalent cations, preferably monoatomic or very low molecular weight, and a stripping polymer, ie, heparin, having multiple anionic moieties. Preferably, these solutions are mixed. The anionic charge of heparin should be large enough to break the salt cross-links and should preferably be equal to or greater than the charge density of the membrane's sodium alginate. A solution is contacted with the capsule, causing the cations, calcium ions, to compete with the polylysine in the membrane for the anionic portion of the sodium alginate in the capsule membrane. At the same time, heparin competes with sodium alginate for cation moieties on the polymer chains. This "softens" or "releases" the capsule membrane. To complete the destruction, the capsule is washed and then exposed to a sequestering agent to remove the calcium ions associated with the sodium alginate. Preferred sequestering agents are chelating agents such as citrate or EDTA ions. If the capsule contains living cells, as is the case in important embodiments, it is preferred to mix the sequestering agent with an isotonic aqueous saline solution.

A preferred sequestering agent is sodium citrate.

The present invention also contemplates a method of encapsulating living cells within a protective environment and later releasing the cells. Thus, the present invention provides what can be described as a package that maintains a live cell culture of any type of drug substance in a sterile, stabilizing environment. The culture fluid can then undergo normal metabolism and even mitosis and be released therefrom at a later time.

It is therefore an object of the present invention to provide a method for encapsulating living cells within a permeable membrane and later selectively disrupting the membrane to release the cells.

Another object of the invention is to provide a method for selectively destroying membranes.

Yet another object of the invention is to disrupt membranes without causing disruption to adjacent living tissue.

Yet another object of the invention is to provide a method for encapsulating and later releasing finely divided substances, liquids and solutions.

These and other objects of the invention will become apparent from the following description of a few specific embodiments.

First, a general explanation will be given.

The selective membrane disruption method of the present invention is carried out on a membrane consisting of a matrix bonded by cross-linking of polylysine and sodium alginate salts. Typically, the membrane has a spheroidal shape and contains the substance to be encapsulated therein. However, the method of the invention can be practiced with membranes of other types than spheroidal shapes.

Although virtually any substance, liquid or solid (compatible with an aqueous environment), can be non-destructively encapsulated and subsequently released by the method of the present invention, as currently contemplated, its A significant utility lies in the ability to encapsulate and later release living systems such as cell cultures. Therefore, the following discussion is primarily limited to a discussion of cell encapsulation and release. Those skilled in the art will be able to adapt the method to the encapsulation of less fragile materials without difficulty.

Next, encapsulation will be explained.

The tissue or cells to be encapsulated are suspended in an aqueous medium suitable for promoting its maintenance and ongoing metabolic processes of the particular cell type involved. Suitable media for this purpose are well known to those skilled in the art, and many are commercially available. The average diameter of the cellular or other material to be encapsulated can vary widely between a few microns to a millimeter or more. For example, mammalian islets of Langerhans typically range from 50 to 200 microns in diameter. Fibroblasts, leukocytes, lymphoblastoids, pancreatic beta,
alpha or delta cells, islets of Langerhans,
Individual cells and tissue fragments, such as hepatocytes or other tissue cells, can be encapsulated as desired. Microorganisms could also be encapsulated, including microorganisms that have been genetically modified by recombinant DNA or other techniques.

The ongoing survival of this species depends on, among other things, the availability of required nutrients, the transfer of oxygen, the absence of toxic substances in the medium and the pH of the medium.
Depends on. Hitherto, it has not been possible to house this type of living organism in a capsule and maintain it in a physiologically compatible environment. The problem is that the conditions required for membrane formation are fatal or harmful to tissues, and prior to the invention of the above-mentioned application, no method of forming membranes that could prolong the life of tissues in a healthy state had appeared. I wasn't there.

However, certain water-soluble substances that are physiologically compatible with living tissue and that can become water-insoluble and form retentive aggregates may form "temporary" structures around individual cells, groups of cells, or tissues. It has been found that it can be used to form "capsules" or protective barrier layers. Such substances are usually added to tissue culture media at concentrations of about 1-2% by weight. From this solution, droplets are formed containing both the tissue and its maintenance or growth medium, the solution becoming immediately water-insoluble and gelling at least in the surface layer. A more permanent membrane is then formed over this shape-retentive temporary capsule. This membrane can later be selectively ruptured according to the invention to release the encapsulated tissue without destroying it. If the materials used to form the temporary membrane permit, the interior of the capsule may be reliquefied after the formation of the permanent membrane. This is done by resetting the medium to conditions in which the medium substance is soluble.

A material that can be used to form temporary capsules is polylysine, which is non-toxic, water-soluble, and convertible into a shape-retentive body by changes in the surrounding ionic environment or concentration. The material also contains multiple easily ionized anionic moieties or carboxyl groups that can react with polymers containing multiple cationic groups to form salt bridges. As will be explained later, this substance is
Allows for the deposition of permanent membranes of selected permeability, including being substantially non-porous to levels of hundreds of thousands of Daltons.

A preferred polyanionic material for forming temporary capsules is sodium alginate, which is an acidic, water-soluble natural or synthetic polysaccharide gum. This substance is commonly extracted from plants and is often used as an additive in various foods. Alginates with molecular weights on the order of 150,000+ Daltons can be used, but because of their molecular size they usually cannot penetrate the capsule membrane that is ultimately formed. To make capsules in which liquid alginates are not trapped, alginates of low molecular weight, for example 40,000 to 80,000 daltons, may be used. In the completed capsule, the alginate can be easily removed from the intracapsular region by diffusion through a sufficiently porous membrane.

This substance contains glycosidic linked sugar chains and the free acid groups are present as sodium salts. When sodium ions are replaced by multivalent ions such as calcium, strontium or aluminum, the liquid water-soluble polysaccharide molecules cross-link to form a water-insoluble, shape-retentive gel. This gel can be resolubilized upon removal of ions by ion exchange or sequestering agents. Although virtually any multivalent ion that can form a salt with the acidic gum is effective, a physiologically compatible ion, namely calcium, is employed.

A typical solution composition includes equal volumes of a suspension of cells in a medium and a 1-2% sodium alginate solution in physiological saline. In this case, a 1.2-1.6% solution was successfully used.

In the next step of the encapsulation process, droplets of the desired size are formed from the tissue-containing sodium alginate solution, sufficient to envelop the tissue to be encapsulated. The droplets are then immediately gelled,
Forms a shape-retaining spherical or spheroidal substance. Droplet formation is performed using well known techniques.

A tube containing an aqueous solution of polyvalent cations, a 1.5% CaCl ₂ solution, is fitted to a stopper that holds the droplet former. The droplet forming device includes an elongated hollow body friction fit to a housing having an upper air intake nozzle and a stopper. A 10 c.c. syringe equipped with a step-actuated pump is mounted on top of the housing, and a Teflon-coated needle, eg, 0.01 inch inside diameter, is threaded through the elongate body of the housing. The interior of the housing is designed so that the tip of the needle is exposed to a constant thin layer of air flow that acts as an air knife. In use, once the syringe is filled with a solution containing the encapsulated substance, the step-actuated pump is activated;
Gradually push out a small drop of solution from the tip of the needle. Each droplet is "cut off" by the airflow,
It falls approximately 2.5 cm into the CaCl ₂ solution, where it immediately gels due to the absorption of calcium ions. In this example, the distance between the tip of the needle and the surface of the CaCl ₂ solution takes the shape that is physically preferable for the sodium alginate and cell suspension, namely spherical shape (maximum volume for minimum surface area). be large enough to hold the Air within the tube escapes through the opening in the stopper. In this way, "cross-linking" of the gel occurs, forming a highly viscous, shape-retentive, temporary protective capsule containing the floating tissue and its medium. The capsules are collected as a separate phase in solution and separated by suction.

In the next step of the process, a membrane is applied around the surface of the temporary capsule by crosslinking the surface layer. This exposes a temporary capsule containing polyvalent anions to an aqueous solution of polylysine, a polymer containing cationic groups reactive with the anionic functional moiety of the polyanionic polymer. In this state, the carboxyl group of alginic acid and the amino group of polylysine are crosslinked by interaction (formation of a salt bridge). Advantageously, permeability can be controlled within limits by selecting the molecular weight of the crosslinking polymer used, varying the exposure time and the concentration of the polymer in solution. A solution of a polymer with a low molecular weight will penetrate more into the temporary capsule than a high molecular weight polymer at a given time. The degree of penetration of the crosslinker was correlated with the permeability obtained. Generally, the higher the molecular weight and the lower the permeability, the larger the pore size. Longer exposure times and higher concentrations of polymer solution tend to reduce the upper limit of permeability of the resulting membrane. However, the average molecular weight of the polymer is the main determining factor. In general, polymers within the molecular weight range of 10,000 to 100,000 Daltons or more can be used, depending on the duration of the reaction, the concentration of the polymer solution, and the desired permeability. Approximately 35000
A successful set of reaction conditions using polylysine with an average molecular weight of Daltons is 0.0167
% polylysine in physiological saline for 3 minutes under stirring. This results in a membrane with a permeability upper limit of about 100,000 Daltons. In general, substances with a high molecular weight form membranes that are later difficult to rupture compared to substances with a low molecular weight.The charge density of the crosslinking polylysine also influences the pore size and the ease of membrane rupture. Generally, materials with higher charge densities form materials with smaller pores and are more difficult to destroy. Optimal reaction conditions suitable for controlling permeability in a given system can be readily determined empirically using the guidelines set forth above.

At this stage of encapsulation, a capsule containing a gel-like solution of sodium alginate, a culture medium compatible with the cells, and a "permanent" semipermeable membrane surrounding the cells can be assembled. If the goal is simply to protect the cells in a protective environment, the subsequent steps need not be performed. However, if mass transfer within the capsule and across the membrane is to be facilitated, it is preferred to liquefy the gel to an aqueous form. This can be done by resetting the conditions under which the sodium alginate is a liquid, for example by removing calcium from the gel. The vehicle within the capsule can be resolubilized by simply immersing the capsule in phosphate buffered saline containing alkali metal ions and hydrogen ions. When the capsules are immersed in the solution under agitation, monovalent ions are exchanged with the calcium in the sodium alginate. Sodium citrate solution can also be used for the same purpose and acts to sequester divalent ions. Sodium alginate molecules having a molecular weight below the permeability limit of the membrane can later be removed from the intracapsular space by diffusion.

Finally, it may be desirable to treat the capsule to immobilize free amino groups or the like.
Otherwise, the capsules tend to clump together.
This treatment can be accomplished by soaking the capsules in a dilute solution of sodium alginate.

It is clear from the foregoing that the membrane formation process does not require the use of harsh chemicals, extreme temperatures or other conditions that are detrimental to cell health and viability. Thus, mammalian hepatocytes,
Even very sensitive cells such as leukocytes, fibroblasts, lymphoblasts and cells from various endocrine tissues can be encapsulated without difficulty. Of course, cells of microorganisms such as yeasts, molds, and bacteria that are well adapted to survive in hostile environments, as well as inert drugs, solids, or biologically active substances, can be encapsulated without destruction.

Encapsulated cells of the type described above can be suspended in a maintenance or growth medium for storage or culture and will be immune to cell infection. Cells undergoing mitosis in vitro do so within the capsule when suspended in a growth medium.
Normal in vitro metabolism continues as long as the components required for the metabolic process are of low enough molecular weight to permeate the capsule membrane or are encapsulated with the cells. Cellular metabolic products (consisting of low molecular weight below the upper limit of permeation) permeate the membrane;
collected within the medium. Cells in encapsulated form can be stored, transported or cultured as desired and released from their protective environment without being destroyed by subsequent steps that selectively disrupt the membrane.

Next, destruction of the membrane will be explained.

According to the invention, the encapsulated material can be released by a two-step process involving commercially available agents that have properties that do not adversely affect the encapsulated cells.

Initially, the capsules are separated from their suspension medium, rinsed to remove contaminants, and dispersed under stirring into separate or preferably mixed solutions of calcium ions and heparin. Heparin, a natural sulfonated polysaccharide, is preferred for disrupting membranes containing cells. The anionic charge of heparin must be sufficient to break the salt cross-links. Thus, the anionic charge density could be equal to, or preferably greater than, the charge density of the sodium alginate originally employed to form the membrane.
In suspension, calcium ions compete for anionic moieties on the sodium alginate with the internal polycations that make up the membrane. At the same time, heparin competes with sodium alginate in the membrane for the cationic moiety of polylysine. This results in a water-dispersible or preferably water-soluble complex of polylysine and heparin polyanions and association of calcium ions with gel molecules.

This step renders the membrane sensitive to subsequent exposure to the sequestering agent. The sequestering agent is 2 or 3 from the gel.
The destruction process is completed by picking up the valence ions. Any capsule membrane debris that normally remains within the medium can be easily separated from the cells.

A preferred solution for the first stage of the selective destruction process contains 1.1% calcium chloride (w/v) and 500-1500 units of heparin per milliliter of solution. A quantity of microcapsules is added to this solution in an amount sufficient to constitute about 20% to 30% of the total suspended solution volume. Calcium chloride and heparin are preferred in disrupting the membrane of the capsule containing the cells. This is because both drugs are physiologically compatible with most cells, minimizing the possibility of cell destruction.

Generally, the concentration of calcium ions and heparin in the solution used in this step can vary widely. Optimal concentrations can be easily determined experimentally. The lowest effective concentration for a particular batch of encapsulated cells is preferred.

The preferred sequestering agent for accomplishing selective destruction is sodium citrate, although other alkali metal citrates and alkali metal EDTA can also be used. If sodium citrate is employed, the optimal concentration is approximately 55mM. If the capsule membrane being disrupted contains viable tissue, the citrate is preferably dissolved in isotonic saline to minimize disruption of the cells.

The invention will be better understood from the following examples.

Capsule formation example 1 Encapsulation of pancreatic tissue Islets of Langerhans were obtained from the pancreas of a mouse, and approximately
were added to a complete tissue culture (CMRL-1969, manufactured by Connaught Laboratories, Toronto, Canada) at a concentration of 10 ³ islets/ml. The tissue culture contains all the nutrients necessary for the continued survival of the islets as well as the amino acids employed by the cells to make hormones. Then, about 10 ³
island / milliliter of Langerhans island floating liquid,
1.2% sodium alginate (Sigma Chemical Company) dissolved in physiological saline.
Add to 1/2 liter of sodium alginate solution.

A 1.5% calcium chloride solution is then used to gel droplets of 300-400 microns in diameter.
After removing the solution on the surface by suction, 2-(cyclohexylamino)ethanesulfonic acid was added to 0.6% NaCl.
15 ml of a solution made by diluting 1 part of a 2% buffer solution (isotonic, pH = 8.2) with ₂₀ parts of 1% CaCl2
Transfer the gelled droplet to the beaker containing the gel. After 3 minutes of soaking, the capsules were washed _twice with 1% CaCl2.

The capsules are then transferred to a 32 ml solution containing 1/80 of a solution of 1% polylysine (average molecular weight 35000 amu) in physiological saline. After 3 minutes, drain the top of the polylysine solution. 1% capsule
Washing with CaCl ₂ followed by an optional step with morpholinopropanesulfonic acid buffer (0.2M, pH
= 6) dilute a 3.3% stock solution of polyethyleneimine with 1/1% _CaCl2 and add 3.3% to a solution of polyethyleneimine (MW40000-60000) prepared to obtain a final polymer concentration of 0.12%. Let this float for a minute. The resulting capsules with a "permanent" semipermeable membrane were washed twice with 1% CaCl ₂ and twice with physiological saline and mixed with 10 ml of 0.12% alginate solution.

The capsules resist aggregation and many are found to contain islets of Langerhans. The gel inside the capsule is liquefied by immersing the capsule in a mixture of saline and citrate buffer (pH-7.4) for 5 minutes. Finally, the capsule is CMLR-
69 suspended in medium.

When viewed microscopically, these capsules are seen to contain a thin membrane surrounding the islets of Langerhans. Individual cells can be seen within the islets of Langerhans. Molecules with molecular weights up to about 100,000 can cross the membrane. This results in
Oxygen, amino acids, nutrients and plasma components used in the culture medium (eg fetal calf serum components) can reach the islets of Langerhans and insulin can be extracted.

Example 2 Encapsulation of Hepatocytes The procedure of Example 1 is repeated, except that 0.5 ml of liver cell suspension containing about 10 ⁵ cells/ml is used instead of 0.4 ml of islet suspension. Liver cell viability was demonstrated by dye exclusion techniques (trypan blue exclusion) and the observed ability to continuously produce urea. Liver tissue in vitro is
It is known that they can ingest toxins from their environment. Therefore, toxins of small enough molecular weight to pass through the semi-permeable membrane are detoxified by the cell.

Example 3 Encapsulation of Activated Charcoal No procedure was carried out except that particulate activated charcoal was suspended directly in sodium alginate solution, 1/2 milliliter of tissue suspension was omitted, and polylysine with an average molecular weight of 35,000 was used as a crosslinking agent. Repeat the same procedure as in Example 1. If the charcoal particles are smaller than the minimum internal diameter of the capillary tube used to create the droplets, a high surface area charcoal surrounded by a semi-permeable membrane results. These particles effectively inhibit the escape of charcoal fragments and dust, but
It can be used to absorb substances of a preselected molecular weight range from fluids passing through the capsule.

Example 4 Encapsulation of human fibroblasts Human fibroblasts obtained by treating human foreskin with trypsin and EDTA for 5 minutes at 37°C in a well-known manner were treated with 40% (v/v) Suspend in complete growth medium (CMLR1969, manufactured by Connaught Laboratories) supplemented with purified fetal calf serum, 0.8% sodium alginate (Sigma) and 200 mg/ml of purified calf skin collagen. The concentration of the cell suspension is 1.5×10 ⁷ cells/ml. A temporary alginate capsule is formed as described above. Capsules made of poly-L-lysine (molecular weight 43,000 daltons)
Attach a semipermeable membrane to the surface layer of the capsule by suspending it in a 0.005% (w/v) aqueous solution of

The resulting capsules are suspended in CMLR-1969 supplemented with 10% fetal calf serum. All steps described above are performed at 37°C. After incubation at the same temperature, microscopic examination reveals that the capsules contain fibroblasts undergoing mitosis and exhibiting a three-dimensional fibroblast morphology within the microcapsules.

Selective destruction of membrane Example 5 Any of the microcapsules of Examples 1 to 4,
The capsule membrane can be selectively destroyed without destroying the encapsulated core material as described below.

A 10 ml portion of the microcapsule suspension containing 500-5000 capsules/ml is allowed to settle and the suspension medium is aspirated. Wash the capsules twice in salt water. The washed capsules were placed in saline solution containing various concentrations of heparin and 1.1% (w/v) _CaCl2 as described below.
Mix with 3.0ml. At the completion of this step, the alginate-containing capsule has a gelled, shape-retaining inner core. The suspension is stirred for 10 minutes at 37°C, after which the capsules are allowed to settle, the surface liquid is aspirated, and the capsules are washed twice with 3.1 ml of 0.15M NaCl. After aspirating the second wash, mix with 2.0 ml of a mixed solution containing equal volumes of 110 mM sodium citrate and 0.15 M NaCl (pH=7.4).

When treated with 1000 units/ml heparin and vortexed for 1 minute in a NaCl-sodium citrate solution, the capsule membrane completely collapsed. The same result is
treatment with 2000 units/ml heparin for 2 minutes, followed by
It can also be obtained by manually vortexing for 15-30 seconds. Low concentrations of heparin are preferred as they reduce the likelihood of cell destruction.

After dissolving the membrane, any membrane debris can be removed by suction and washing. After resuspending the released cells in culture medium, they can be tested by the tryptan blue dye exclusion technique, which shows that the cells are in a healthy viable state and exhibit relatively little dye uptake. Let's understand.

Reference Example 1 Capsules manufactured according to Example 3 were
After washing, 1000 units/ml heparin and 1.0%
Treat with 3.0 ml of a solution containing AlCl ₃ for 6 minutes with stirring. After aspirating the surface liquid, the core material is released by vortexing the capsule in a 0.1 M solution of sodium citrate for 30-90 seconds.

Reference example 2 At pH 7.0 instead of sodium citrate
Using 0.10M EDTA (sodium form)
The procedure of Reference Example 1 can be repeated, except that rapid rupture of the capsule membrane occurs.

Reference Example 3 Capsules manufactured according to Example 3 were prepared after washing and containing 10 mg/ml polyvinyl sulfate (molecular weight approximately 50,000 Daltons) and 1% CaCl ₂
Treat with 3.0 ml of aqueous solution. After treatment with 0.10M sodium citrate, the capsules were virtually completely dissolved.

Other embodiments are possible within the scope of the claims.

Claims (1)

Claims: 1. A method for encapsulating a core material, which is an individual cell, tissue fragment, or microorganism, in a protective environment and subsequently releasing the core material, comprising: (A) an aqueous medium containing sodium alginate; (B) forming droplets of the medium; (C) exposing the droplets to a calcium chloride solution to gel the droplets as discrete, shape-retentive, water-insoluble capsules; , (D) crosslinking the surface layer of the capsule to form a semipermeable membrane around the droplet by exposing the droplet to polylysine. encapsulating the core material and (E) exposing the capsules obtained from step D to a solution of calcium chloride and a solution of heparin; (F) sequestering the calcium ions associated with sodium alginate after step E; A method comprising: releasing said core substance by steps comprising: 2. The method of claim 1, wherein said capping step F is carried out by exposure to a solution containing said capsule chelating agent. 3 The chelating agent contains citrate ion and
3. The method of claim 2, wherein the EDT ion is selected from the group consisting of EDT ions. 4. The method according to claim 1, wherein the capsule is a microcapsule containing living cells. 5. Claim 4, wherein said blocking step is carried out with said cells and citrate dissolved in physiological saline.
The method described in section.