.' The present invention relates to .culturing of 5 -cartilage cells _in vitro to produce cartilage tissue. vTlie resultant tissue and its growth medium has utility - .as: a-. :so_ur.ce of a protein fraction having anti-invasive prope ties. .Certai .tissues, have a high resistance to 10 invasion, by foreign cells, and cartilage tissue has been found .to have= especially -high resistance to invasion by .fo eig -reells such as cancer cells and blood capilliαry cells.

.Described in- my U.S. Patent" o. ^• 4,042,457, 15 issued August 16, 19 ^' 77,. is a protein fraction obtained .from cartilage tissues which inhibits. cell proliferation and: ti.ss.ue .invasion. This protein fraction was also ^" -found- to have, inhibitory effects on bone resorption- as ^• .described in my U.S. Patent No. 4,176, 77, issued 20 November: 27, 19.79. This protein fraction will hereinafter be referred to as anti-invasion factor, or

AIF K s^ ^' bfe.en- ^* shown ^v to ^{1 '} exp^es's ariti-tumor

...properties both in vivo and in vitro. Tumors grown in

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25 injected with sufficient AIF demonstrate regression of . .

^■ the tumors and a la k ^' of new daughter tumors. ' ^'" " ^" '_ ^'

. Accordingly, AIF holds promise as a cancer- ^' treat ng agent. However, AIF must bes made more readily - ...-,.- ,

30 obtainable for its full drug potential, to be. realized.

Current methods of obtaining AIF involve ^' obtaining waste animal parts from slaughterhouses, separating the cartilage tissue and e tr cting_the AIF.

This is a long and tedious procedure. Cartilage tissue

-2- is a part of the skeleton of animals and, accordingly, bones and adjacent muscles and other connective - ssu s ^" must be separated to reach the cartilage to r.eπ^^..th small amount of cartilage tissue therefrom. -...-'- ---,. - As a first step toward increasing th ^ay^ilable amount of AIF, it would be desirable to obtain..cart lage tissue by means other than separating animal .joints _£r , such as by culturing the cartilage cell population.-,...--, chondroblasts, or chondrocytes to obtain cartilage tissue having significant amounts o AIF. -_ _; -• '. Culturing of cartilage cells containing .. _rπ significant amounts of AIF is complicated by the.,,.... . inherent nature of cartilage tissue. Cartilage cells in vivo produce an extracellular matrix which separates one cell from another. One of the main functions of-the - cells comprising cartilage would appear to be the. _ .. production of the extracellular matrix. These cartilage forming cells are called ^' chondroblasts. While an animal is growing, the cartilage is growing and many.cartilage cells retain their ability to reproduce. _. ■

In an adult animal, the majority of cartilage cells are embedded within the extracellular matrix and are non-reproductive and responsible for the maintenance of the tissue integrity. Even though functionally incorrect, all cartilage cells, whether reproduetively active or inactive, are generally called chondrocytes^ The extracellular matrix, comprised largely of collagen type II and proteoglycans is extremely long lasting, cartilage collagen in certain animals may have a. half-life of several years. Accordingly, when the chondrocytes have produced the extracellular matrix around themselves and thereby produced the cartilage tissue, they have generally served their function. Chondrocytes from adult cartilage tissue have generally lost their in vivo potential to reproduce.

-3-

^■ . ^* i-.?- ^• * ^> >-' he most actively reproducing chondrocytes are ^■' "t ^' σ^r ^~ fdlα i? in embryonic tissue. However, embryonic tiVsue' ^c a£ipears to contain less anti-invasion factor than adolescent tissue. Illustrative of this fact is that while'- aB/ cartilage tissue is nonvascular, as a result σf--t:he aniti-invasion factor preventing the ingrowth of capii-i-ary- cells, some early embryonic cartilage is supplle-d ^'1 >y the vascular system.

^£:" - ^L ft further well recognized difficulty in culturing cartilage tissue is the fact that chondrocytes do not necessarily produce cartilage tissue when culture' v ~- - ^{': : '} ϊien chondrocytes obtained from most cartilage tissue ^{1 "} are cultured, the resulting tissue includes fibrobla ^' sts, that is., cells which have not differentiated into cartilage producing cells or modulated chondrocytes with fibroblastic gene expression. Such cultures produce collagen type I, the most ^" -common ^1' -type of collagen which is found in tissues such as skin, bone, muscle, etc., as well as collagen type TT, the type of collagen found in cartilage. In order to produce true cartilage tissue, therefore, it is necessary that the chondrocytes maintain their differentiated state and proliferate only into cartilage type ceils with an extracellular matrix therearound containing only collagen type II. While collagen type I -' would appear not to be a component of AIF, the production of collagen type II is a good indicator of the presence of AIF as both collagen type II and AIF are closely associated with cartilage tissue.

It has been recognized that relative amounts of collagen type I and collagen type II which have been obtained from chondrocyte cultures depend on the environment surrounding the cultured chondrocytes. Different cellular environments give rise to cultures

with collagen type I and collagen type II in varying amounts. An extremely significant environmental factor has been shown to be the density in which the chondrocytes are suspended and plated. For uncertain reasons, chondrocytes resist contact with other chondrocytes and when chondrocytes are in contact with other such cells, they produce around themselves the extracellular matrix found in cartilage. Studies with embryonic chicken chondrocytes have shown that when the chondrocytes are plated in very high density, primarily collagen type II is produced. Likewise, it has been found in fetal bovine cartilage tissue that a high density of plated chondrocytes yields a high percentage of collagen type II as compared to collagen type I. The plating of mature mammalian chondrocytes to produce cartilage tissue heretofore has been less than completely successful in that culturing of mammalian tissue has generally produced cultures having a mixture of collagen type I and collagen type II. in order that anti-invasion factor may be produced in greater amounts it would be desirable to be able to culture mammalian chondrocytes to produce cartilage tissue. To insure sufficient yield of anti-invasion factor, it would be highly desirable to culture chondrocytes to produce cultures with extracellular matrices containing only collagen type II.

Accordingly it is an object of the present invention to culture mammalian chondrocytes to produce cartilage tissue with high amounts of anti-invasion factor.

The objects are achieved in the present invention by plating, in high density, chondrocytes obtained from mammals which have a high percentage of viability so that, generally, each active chondrocyte is in surface contact with another active chondrocyte.

While it has been recognized that high density plating tends to produce cartilage tissue, previous attempts have been less than successful due to the failure to concentrate reproductively viable cells in sufficient density.

To more fully understand the present invention, the culturing of chondrocytes to produce cartilage tissue will now be described in greater detail.

As hereinabove described, the most active cartilage tissue, i.e. embryonic tissue, is limited in the production of anti-invasion factor, and mature cartilage is metabolically slow and contains cells which are generally inactive, and therefore, when mature tissue is degraded by the best known methods a very low yield of viable chondrocytes is provided. It is therefore desirable to obtain cells from non-embryonic animals which nevertheless provide a high percentage of viable chondrocytes.

It has been found that the chondrocytes of weight bearing articular cartilage as are found in the ankles and toes of adolescent mammals have a very high viability and high degree of biological activity. (The term "adolescent" is used herein in its medical sense to refer to the entire growing period of the animal from birth to maturity.) In particular, calf fetlock cartilage contain a high percentage of reproductively and metabolically active chondrocytes. A growing calf gains weight very rapidly, and to maintain the calf's weight, the weight bearing limbs such as the hooves and fetlocks must be enlarging proportionally to accommodate the increasing weight, and the cartilage tissue is proportionally growing and expanding.

Calf fetlocks are generally a waste product of slaughterhouses and may therefore be obtained with relative ease. After the -animal is slaughtered, the

fetlocks are transported to the laboratory. The skin is removed and the first phalangal joint is carefully ^• opened under aseptic conditions and the synovial fluid is removed. The cartilage of the exposed joint is carefully shaved off so as to obtain as much cartilage tissue as possible and yet avoid contamination of other tissue ^' s such as bone. The shavings are collected in Ham's F-12 medium or any other tissue culture medium suitable for tissue survival but enriched in antibiotics and antifungal agents, i.e. 50 micrograms gentamycin and 5 micrograms of beta-amphotericin per milliliter. The cartilage shavings contain both the chondrocytes and the extracellular matrix. In order that the cells may be cultured, the extracellular matrix must be removed. This is done enzymatically by various enzymes which digest the proteins of the extracellular matrix. The shavings are first digested with pronase in an amount between about 0.5 and about 2.0% and most preferably about 1% weight per volume in Ham's F-12 medium or other suitable tissue culture medium containing 5% bovine serum for- 90 minutes. The pronase will digest most of the proteins of the extracellular matrix including proteoglycans and some collagen. Therefore, the shavings after washing and centrifugation are further digested in medium containing 5% bovine serum and between about 0.1 and about 1% weight per volume collagenase for a period of about 1-2 hours to digest the collagen from around the cells. The cells, after washing and centrifugation, are further digested with between about 0.1% and about 3% weight per volume trypsin or testicular hyaluronidase and most preferably about .25% trypsin or hyaluronidase for between about 5 and about 10 minutes in the presence of 5% bovine serum. The trypsin will digest any remaining extracellular proteins to denude the cells. Testicular _

hyaluronidase will digest the sugar moieties attached to any remaining extracellular protein and. thereby denude the chondrocytes.

The digestion of the extracellular material by the proteolytic enzymes is very harsh to the cells. The enzymes, in addition to attacking the matrix, begin to attack ^' the cell membranes. Accordingly the cells are left in a weakened state after digestion. Mammalian serum, preferably fetal serum, is therefore added to the ^" digestive mixture so that the denuded cells may be coated and protected with serum immediately after being denuded.

The fact that serum and digestive enzymes can be used simultaneously is somewhat surprising for several reasons. It would be expected that all the trypsin would attack the serum and would bind, for the most part, with proteins of the serum. Furthermore, serum contains trypsin inhibitors which should affect trypsin's action vis a vis the extracellular matrix. Nevertheless, it has been found that this combination of trypsin along with serum produces chondrocytes with a high percentage of viability.

The isolated cells are collected by centrifugation at about 900 rpm for 10 minutes. The cell pellet is washed in phosphate buffered physiolo ical saline pH 7.4 as described in J. Cell Biol. 49: 451, (1971) and the cells are suspended in Ham's F-12 medium without serum. The cells are then passed through a 90 micrometer Nitex screen to separate the cells which are resuspended in Ham's F-12 medium containing about 5-10 percent serum. Sufficient cells are added to the medium to achieve a concentration of at least 10 cells per milliliter.

Cells may be grown in culture dishes such as 35 mm tissue culture dishes and for morphological

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observation may be grown on 26 x 34 mm cover slips submerged in culture dishes.

In order to produce cartilage tissue, at least a monolayer of denuded chondrocytes must be originally plated. Most of these cells must be reproductively viable in order for growing chondrocytes to contact other growing chondrocytes to produce the extracellular matrix. Accordingly, cartilage tissue which will provide at least about 60% viable chondrocytes, is selected for culturing. Herein, the percent of viable chondrocytes referred to is that percent of chondrocytes which is provided by careful but thorough degradation of the extracellular matrix of a particular tissue.

It has been found that chondrocytes obtained from fetlock cartilage of adolescent bovines may be 89% reproductively viable. This is somewhat surprising in light of the viability of chondrocytes obtained from other tissue such as nasal cartilage from which, because of its easy accessibility, cartilage tissue is commonly obtained. For example, in the same animal where 89% reproductively viable chondrocytes may be obtained from fetlock cartilage degradation, only 10 to 15 percent reproductively viable chondrocytes may be obtained from nasal cartilage degradation. It is therefore possible to plate reproductively viable fetlock-derived chondrocytes in sufficiently high density to produce at least a monolayer of viable cells, such a density being necessary to produce cartilage tissue.

While chondrocytes grown in culture dishes produce the desired tissue, significant amounts of tissue cannot thereby be produced. Culture dishes are small. Furthermore, an important factor in achieving good yields of cartilage tissue is the atmosphere in which the chondrocytes grow. After layers of cells begin to accumulate, the lower layers are shielded from

the atmosphere. Inability to maintain optimal gas balance for the shielded layers results in reduced production of cultured material.

In order to produce cultured material in large quantities, it is desirable to use roller bottles. The use of roller bottles in tissue culture involves plating cells on the interior surface of a cylindrical container, providing the cells with an appropriate growth medium and rotating the container while the cells grow. The application of culturing techniques to roller bottle technology is necessary if a culture is to be mass produced. Roller bottles have large surface areas and permit easy exchange of the medium and gases. Because of the sensitivity and difficulty of culturing chondrocytes, roller bottle technology was heretofore considered unsuitable for the culturing of cartilage tissue. However, it has been found that good growth of cartilage tissue may be achieved in roller bottles from cells produced according to the above methods. The cultures are maintained in Ham's F-12 medium supplemented with the antibiotic and antifungal agents described above as well as with 50 micrograms of ascorbic acid per ml. and 15% bovine serum. The cells are cultured at 37° in a humidified atmosphere containing at least 5% carbon dioxide. The medium is changed every other day. After about 10 days, nodules appear which contain extensive extracellular matrix chemically identified as being cartilaginous in nature. AIF may be extracted from the chondrocyte cultures as well as the collected growth media according to the methods described in my Patent No. 4,042,457.

Example 1 o

About 10 chondrocytes are prepared and cultured by the above described method in a roller bottle having 500cm ² of interior surface to achieve a

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5 2 plated density of about 2 x 10 cells per cm of interior surface area. After 16 days the culture, which

Q contains an estimated 3 x 10 cells has an extracellular matrix which morphologically and biochemically resembles the extracellular matrix of the adolescent bovine cartilage tissue from which the chondrocytes are obtained.

Example 2 2 x 10 chrondrocytes are prepared and cultured by the above described method in a 35 mm culture plate (a density of plated cells of about 2 x

1 1(0 5 cells/cm2) for 16 days in the presence of 3, H-proline (10 micro Ci/ l) . The scraped cells and medium combined are brought to 25% saturation with ammonium sulfate and the precipitate is collected by centrifugation following an overnight incubation at 4°C. The pellet is dissolved in 0.5 M acetic acid containing 1 mg/ml type I carrier collagen and 10 micrograms per ml. pepsin. Digestion is carried out for ιo hours at 4°C. The collagen mixture is brought to pH 8 and centrifuged to remove undigested material. The supernatant is again brought to 25% saturation with ammonium sulfate to precipitate the protein. The pellet produced by centrifugation is dissolved in 0.5 M acetic acid and exhaustively dialyzed against the same solution. The collagen, free of labeled unincorporated amino acid is lyophilized and subsequently dissolved in 6 M urea with 1% SDS.

The collagen is denatured at 70° C for 30 hours and electrophoresed under normal and reducing conditions according to Laemli, Nature, 227, 680, (1970) .

A 5% running gel separates the alpha 1 chains of type II collagen from the alpha 2 chains of type I collagen and the ratios of labeled alpha 1 to alpha 2 chains is determined. The band containing the alpha 2

/

chains of the carrier type I collagen contains no label while the band containing alpha 1 chains is highly labeled indicating the production of only collagen type II by the culture. While the invention has been described according to the best mode presently known to the inventor, modifications obvious to one skilled in the art may be made without departing from the scope of the present invention which is limited only by the following claims: