WO2002092794A2 - Isolation method of mesenchymal cells - Google Patents

Abstract

The invention relates to a method for isolating mesenchymal cells from dermal tissue comprising the steps of : - incubating dermal tissue in a buffer containing collagenase type II to produce a digest; - filtering the digest; and - centrifuging the digest.

Description

HARVESTING OF CELLS

The invention relates to a process of harvesting cells from living tissue. In particular, the invention relates to a process for harvesting living mesenchymal cells from dermal tissue.

There is a current trend in medicine towards the use of tissue engineering as a technique to repair or even substitute damaged tissue. Recent developments encompass the design of tissue engineered bone, cartilage, and skin. In the future, it is envisages that even complete organs, muscle and nerve tissue may be treated using tissue engineering protocols.

Simply put, tissue engineering is a technique in which living tissue is produced in vitro and is incorporated into a patient's body to replace or supplement the same type of tissue there. Typically, the technique comprises selecting suitable cells and culturing them to produce the desired tissue. The cells chosen may be of the same type as the tissue of the end product, however, often different, e.g. less developed cells such as stem cells, are used. The culturing thus comprises both proliferation and differentiation of cells.

In many cases, in particular in the field of bone and cartilage tissue engineering, use is made of a scaffold to culture the cells on. This scaffold is intended to hold the cells together and provide the tissue engineered product with sufficient mechanical integrity and strengths to allow it to be implanted. It is usually preferred that the scaffold is of a biodegradable material. This has the effect that the scaffold material slowly degrades and is removed through natural processes as the implanted, in vitro produced tissue adjusts to its living surroundings and gradually takes over the scaffold's tasks.

An important aspect of all types of tissue engineering is the avoidance of any adverse reactions of a patient to the implanted tissue engineered product. Accordingly, it is of the utmost importance not only that the tissue engineering process is carried out aseptically to prevent microbial contamination, but also that proper biocompatible materials are selected to avoid rejection of the tissue engineered product. With respect to the selection of cells, this implies that it is highly desired to use autologous cells to produce a tissue engineered product from.

The use of autologous cells means that tissue engineered products must be specially manufactured for each different patient. Usually, it further means that cells must be harvested from a patient who is to receive a tissue engineered product first, before the tissue engineering can be started. It will be understood that it is in the patient's interest that the time required between the harvesting of cells and the implantation of a tissue engineered product is as short as possible. Because patients in need of tissue engineered skin generally suffer from wounds, such as burns and the like, that need immediate attention and treatment, in the field of tissue engineering skin it is particularly important that the procedure from the harvesting of cells through the tissue engineering process to the treatment of the patient with the in vitro produced skin substitute takes as little time as possible. Clinicians generally prefer to treat their patients' wounds as quickly as possible. With respect to the harvesting of the cells, it is further desired that the yield of suitable cells from a skin biopsy taken from the patient is as high as possible. The more cells can be harvested from a skin biopsy, the smaller the biopsy can be and the less discomfort the patient will suffer from.

In the past, isolation techniques used for obtaining cells from dermal tissue taken from a patient vary from explant cultures to digestion of the tissue with a protease to yield a cell suspension that can be cultured immediately. However, it has been found that the cell yields using these techniques are not sufficient to create large autologous skin substitutes.

The present invention provides a method for isolating cells to be used in a process for producing tissue engineered skin substitutes from a skin biopsy that does not suffer from the disadvantages of the known methods. In particular, a method is provided which can be carried out quickly and easily to minimize the time between the taking of a skin biopsy and the treatment of the patient with the in vitro produced skin substitute. In addition, in the present method a high yield of cells from a skin biopsy is achieved to allow the production of large autologous skin substitutes from relatively small biopsies. A method according to the invention comprises a number of specific steps that lead to the mentioned advantageous results. Thus, the invention specifically relates to a method for harvesting mesenchymal cells from dermal tissue comprising the steps of:

- incubating dermal tissue in a buffer containing collagenase type II to produce a digest; - filtering the digest; and

- centrifuging the digest.

As mentioned, in accordance with the invention mesenchymal cells are harvested from dermal tissue. Mesenchymal cells are cells derived during embryogenesis from mesoderm e.g. fibroblasts, smooth muscle cells or pericytes. Mesenchymal cells stain specifically positive for the protein vimentin. These cells have found to be particularly useful for tissue engineering skin.

The dermal tissue will generally be obtained from a skin biopsy taken from a patient who is later to be treated with a tissue engineered product prepared from the mesenchymal cells harvested from said dermal tissue. The skin biopsy may be taken in any known manner from any suitable location on the body of the patient. Typically, biopsies will be taken from the upper thighs, buttocks or upper arms of the patient's body. The skin biopsy will usually be transported for a certain period of time, preferably as short as possible. This is preferably done at low temperature, such as from 0-10°C in suitable medium. An example of a suitable medium is RPMI (Roswell Park Memorial Institute) Media 1640. In a preferred embodiment, antibiotics, such as gentamycin, penicillin and/or streptomycin, are added to the medium to avoid microbial contamination of the biopsy. After storage, the skin biopsy may suitably be divided into small pieces to facilitate their further processing. As a typical example, the skin biopsy is cut into pieces of 5x5 mm. Subsequently, these pieces are preferably incubated in a buffer containing dispase grade II. Suitable buffers include phosphate buffered saline (PBS) pH 7.2-7.4 and Tris-HCl pH 7.2-7.4 having an osmolarity similar to the human body fluid (300-320 mmol). Dispase II is isolated from bacillus poly my x and commercially available from e.g. Roche diagnostics. The dispase II is preferably present in the buffer in an amount of at least 0.1% (w/v). As increasing its amount essentially does not increase its advantageous effects, the dispase II will usually not be employed in amounts greater than 1% (w/v). In a much preferred embodiment, the dispase II is used in an amount of 0.2-0.3% (w/v).

The incubation in the buffer with the dispase II preferably lasts between 20 and 60 minutes, more preferably between 30 and 45 minutes. The temperature at which the incubation is carried out is chosen to resemble body temperatures. Accordingly, it will generally lie between 36 and 39°C.

After the incubation has proceeded for sufficient time, the epidermis that will be present in the skin biopsy will be removed to yield the desired dermal tissue. The removal of the epidermis may be performed in any known manner, for instance using fine forceps.

The thus obtained dermal tissue is incubated in a buffer containing collagenase type II. This incubation step encompasses digestion of the dermal tissue. In this regard, the term 'digestion' is used to indicate enzymatic cleavage of tissue surrounding the mesenchymal cells, especially interstitial collageneous tissues, which can be measured by weighing the decrease in wet- weight of the tissue. After the incubation, a product is obtained which is referred to as a digest, reflecting the digested state of the dermal tissue.

Suitable buffers for the incubation include PBS pH 7.2-7.4 and Tris- HCl pH 7.2-7.4 having a osmolarity similar to the human body fluid (300-320 mmol). Collagenase type II is preferably isolated from Clostridium Histolyticum and defined by having average amount of collagenase, caseinase and tryptic activity and a higher clostripain activity in comparison to collagenase type I, III and IV. These types of collagenase are commercially available from e.g. Roche Diagnostics, Worthington Biochemical. It has been found that collagenases type II has especially high dermal tissue digestion capacity.

Suitable concentrations for the collagenase type II m the buffer range from 0.1 to 3% (w/v) and are preferably from 0.5 to 1% (w/v). The resulting buffer solution containing the collagenase type II will preferably be employed in an amount from 3 to 15, more preferably from 8 to 12 ml per gram of dermal tissue.

The collagenase, clostripain and tryptic activities of collagenase II products widely vary from batch to batch and from supplier to supplier. It has been found that the digesting process can be improved by using collagenase type II that has been selected upon its clostripain activity, its tryptic activity, and/or its collagenase activity. In particular by using a collagenase type II with a collagenase, a clostripain and /or a tryptic activity in a selected range, the dermal tissue digestion capacity can be improved Such a collagenase type II may be obtained by selecting a suitable batch from the activity data that are provided or measured, or by blending two or more batches to obtain a collagenase type II batch with activities in a desired range.

Particularly good results have been achieved with a Collagenase type II having a clostripain activity in the range of about 6-12 U/mg dry weight, a collagenase activity in the range of about 250-500 U/mg dry weight, and a tryptic activity in the range of about 0 2-0.5 U/mg dry weight The collagenase activity herein is measured by liberation of L-leucme equivalents from collagen (1 U = 1 μM L-leucme liberated during a 5 hour incubation at 37 °C), the clostripain activity is measured herein by hydrolyzmg N-α-benzoyl-L- arginine ethylester (BAEE) (1 U hydrolyzes 1 μM BAEE m 1 mm at 25°C at pH 7 6 m the presence of ImM calcium acetate and 2,5 mM dithiotreitol), the tryptic activity herein is measured by digestion of BAEE substrate. (1 U hydrolyzes 1 μM BAEE in 1 min at 25°C at pH 7.6).

The dermal tissue is preferably incubated in the buffer containing the collagenase type II for a time period of at least six hours, more preferably 6 to 24 hours, even more preferably 8 to lOh to achieve a sufficient extent of digestion of the dermal tissue. The incubation is preferably chosen to resemble body temperatures. Accordingly, it will generally lie between 36 and 39°C.

In a preferred embodiment, the incubation is performed in a rotating container. It has been found that rotation has a positive effect on the digestion of the dermal tissue. In principle, any type of rotating container can be used, such as rotating shaker. Preferably, the container is rotated at 30-80 rpm, more preferably at 50-60 rpm. By rotating in this specific manner a high cell viability is achieved.

After the incubation has been completed to a sufficient extent, the digest will be filtered to remove non-digested tissue fragments from the solution. Particularly good results and high cell yields have been obtained by filtering the digest on a membrane having a pore size between 300 and 700 μm, preferably between 400 and 600 μm. Preferably, the filter is washed after the filtration with excess culture medium to optimize the cell yield. Although in principle any conventional culture medium can be employed, good results have been obtained using Dulbecco's Modified Eagle Medium (DMEM). In a preferred embodiment, the culture medium also contains foetal calf serum (FCS, e.g. in a 10% solution) and antibiotics, such as gentamycin, penicillin and/or streptomycin. The filtered digest is then subjected to centrifugation to obtain pellets containing the objective cells. It has been found that cell yields can be increased by carrying out centrifugation at 300-500 g for 5-20 minutes, preferably 380-400 g for 8-12 minutes.

If desired that cells present in the obtained pellets may be resuspended in a culture medium for purposes of further processing, such as proliferation and/or differentiation to produce a tissue engineered skin product.

The invention will now be further elucidated by the following, non- restrictive examples.

Example 1

Skin biopsies were stored for 16 hours at 4°C in Dulbecos Modified Eagle medium containing antibiotics. The skins were cut into small pieces 5x5mm and incubated in a PBS solution containing dispase grade II (0.^v25% w/v) (Roche Molecular Biochemicals) for 30 or 45 minutes at 37°C. The epidermis of each piece was removed using fine forceps.

Each dermal tissue was minced in smaller pieces using scissors and placed in a PBS solution containing a collagenase type II (Life technologies) (0.5 or 1% w/v;10 ml/g of tissue) (Collagenase activity 271 U/mg dryweight, Clostripain activity 6.9 U/mg dryweight, and tryptic activity 0.35 U/mg dryweight). Each tissue was incubated for at least 10 hours at 37°C on XYZ rotating shaker (50 or 60 rpm). Each digest was filtered over a membrane (pore size 400-600μm) and each filter was washed with excess culture medium (Dulbecos Modified Eagle medium containing 10% FCS and antibiotics). Each digest was centrifuged (400g 10 min) and cells in the pellet were resuspended in culture medium, counted and plated on tissue culture polystyrene at a density of 10.000 cells per square cm. The cell yield from several donors (n=7) ranged from 9.7 till 25.7 million mesenchymal cells per gram of dermal tissue. The cell survival was investigated by counting the number of attached cells per square cm 16 to 24 hours after seeding. The cell survival ranged from 60- 87% (n=7).

In conventional methods, using thermolysin or trypsin for a short period at 37 °C or overnight at 4 °C, the cell yield and viability of less than 5 million cells/g tissue may be achieved. In comparison to such methods the method shown in this example clearly shows a significant improvement, (see Normand J, Karasek MA, A method for the isolation and serial propagation of keratinocytes, endothelial cells, and fibroblasts froma single punch biopsy of human skin, In Vitro Cell. Dev. Biol.-Animal. 31:447-455, 1995; Hentzer B, Kobayasi T, Enzymatic liberation of viable cells of human skin, Acta Dermatol.Venerol. (Stockholm) 58: 197-202, 1978.)

Example 2

Several biopsies of several donors were digested as follows:

1. Tissue was washed 3 times with PBS, the tissue was placed in petri dishes and blood coagulates were removed as much as possible by rubbing with foreceps or cutting off tissue parts penetrated by blood (e.g. vascular structures). 2. Epidermis was removed with a thin layer of papillary dermis with a dermatome set at 0.2 mm thickness. The dermal tissues was cut into small pieces with a pair of scissors or with surgical knifes. 3. The tissue fragments were collected in a pre-weighed tube and the weight of the tissue was measured. 4. Several digestion solutions containing collagenase type II (0.4 % w/v in PBS) were prepared freshly and 3 ml per gram of tissue were added.

5. The tubes were closed firmly and sealed with parafilm before placing it in the water bath set at 37°C while gently shaking (65-70 shakes per minute).

6. After 6h, each digest was sieved with an open filter chamber; the filter chamber was washed with 20 ml of fibroblast culture medium (Dulbeco's

Modified Eagle Medium containing 5% Foetal Bovine Serum and the antibiotics penicillin (100 units/ml) and streptomycin (100 μg/ml)).

7. Each digest and washings was centrifuged at 400g for 10 min at room temperature; the supernatant was removed and the pellet was resuspended in the same fibroblast culture medium . 8. Three different ahquots were taken and the large cells excluding granulocytes and red blood cells were counted using haemacytometer.

Six collagenase batches with different proteolytic activities as determined by the manufacturer were tested. The activities and results for each batch are shown in Table 1-

Table 1

Batch Collagenase Clostripain Tryptic activity number of cells number of activity activity (U/mg dry wt ) (cells/g tissue) different

(U/mg dry wt ) (U/mg dry wt ) tissues

I 1037 1.43 0.027 <lxlOE6 5

II 271 4.7 0.46 0.5-2.5xl0E6 7

III 358 10.9 0.47 2-7xlOE6 4

IV 271 6.9 0 35 1.5-5xlOE6 7

V 2330 0.91 0.35 <0.5xlOE6 2

VI 396 6.9 0.09 0.5-1.5xlOE6 4

Batch III and IV yielded the highest numbers of cells after a 6h digestion of the dermal tissue. The combined collagenase, clostripain and tryptic activity was high in these collagenase batches when compared to the other batches m which clostripain or tryptic or both these activities were lower than 6 and 0.2 units/mg dryweight, respectively.

Example 3

A tissue was treated similar to the method described in Example 2, with a digestion solution containing 0.4 % (w/v) of collagenase type II (Life Technologies). A tube containing the tissue fragment and the digestion solution was placed in the water bath (at 37 °C) Samples were taken from the tube, after 2,4,6,8 and 10 hours as described under step 6 in the procedure of Example 2. Cell counts were made for each sample.

The results are shown in Figure 1. Figure 1 clearly shows that in time the cell yield from dermal tissue increased significantly and that in this example the optimal incubation time with the collagenase digestion solution was between 6 and lOh. After plating of the cells in culture, the cell survival was in between 50 and 70% for all digestion times, estimated after 24 h.

Claims

Claims

1. A method for harvesting mesenchymal cells from dermal tissue comprising the steps of:

- incubating dermal tissue in a buffer containing collagenase type II to produce a digest; - filtering the digest; and

- centrifuging the digest.

2. A method according to claim 1, wherein the incubation lasts at least six hours.

3. A method according to claim 1 or 2, wherein the incubation is carried out at a temperature between 36 and 39°C.

4. A method according to any of the preceding claims, wherein the incubation is carried out in a rotating container.

5. A method according to claim 4, wherein the container is rotated at 30-80 rpm, preferably 50-60 rpm.

6. A method according to any of the preceding claims, wherein the collagenase type II is obtained from Clostridium Histolyticum.

7. A method according to claim 6, wherein the collagenase type II is selected upon its clostripain activity, tryptic activity, and/or collagenase activity.

8. A method according to claim 7, wherein the collagenase type II has a collagenase activity in the range of about 250-500 U/mg dry weight, a clostripain activity in the range of about 6-12 U/mg dry weight and a tryptic activity in the range of about 0.2-0.5 U/mg dry weight.

9. A method according to any of the preceding claims, wherein the digest is filtered on a membrane having a pore size between 300 and 700 μm, preferably between 400 and 600 μm.

10. A method according to any of the preceding claims, wherein centrifugation is carried out at 300-500 g for 5-20 minutes.

11. A method according to any of the preceding claims, wherein the dermal tissue is obtained by incubating a skin biopsy comprising an epidermis and dermal tissue in a buffer containing dispase grade II and removing the epidermis.

12. Mesenchymal cells obtainable by a method according to any of the preceding claims.