U.S. patent application number 13/403167 was filed with the patent office on 2012-08-30 for method for the production of intervertebral disk cell transplants and their use as transplantation material.
This patent application is currently assigned to CO.DON AG. Invention is credited to Olivera JOSIMOVIC-ALASEVIC, Jeanette Libera, Hans-Joerg Meisel, Vilma Siodla.
Application Number | 20120219533 13/403167 |
Document ID | / |
Family ID | 34681804 |
Filed Date | 2012-08-30 |
United States Patent
Application |
20120219533 |
Kind Code |
A1 |
JOSIMOVIC-ALASEVIC; Olivera ;
et al. |
August 30, 2012 |
METHOD FOR THE PRODUCTION OF INTERVERTEBRAL DISK CELL TRANSPLANTS
AND THEIR USE AS TRANSPLANTATION MATERIAL
Abstract
The invention relates to a method for the in vitro production of
intervertebral disk cartilage cell transplants from affected
intervertebral disk tissue from patients and to the use thereof as
transplantation material for the treatment of affected
intervertebral disks. The invention also relates to a
three-dimensional, vital, and mechanically stable intervertebral
disk cartilage tissue and to the use thereof as transplantation
material for the treatment of affected intervertebral disks and in
testing active substances. Furthermore, the invention is directed
to the surgical technique for incorporating the transplants, to the
intervertebral disk cell transplants and intervertebral disk
cartilage tissues produced, and to therapeutic formulations, e.g.
injection solutions, which include said tissue and said cell
transplants.
Inventors: |
JOSIMOVIC-ALASEVIC; Olivera;
(Berlin, DE) ; Libera; Jeanette; (Berlin, DE)
; Siodla; Vilma; (Kleinmachnow, DE) ; Meisel;
Hans-Joerg; (Berlin, DE) |
Assignee: |
CO.DON AG
Teltow
DE
|
Family ID: |
34681804 |
Appl. No.: |
13/403167 |
Filed: |
February 23, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10596357 |
Jun 12, 2008 |
8148150 |
|
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PCT/DE2004/002761 |
Dec 13, 2004 |
|
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13403167 |
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60544315 |
Feb 17, 2004 |
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Current U.S.
Class: |
424/93.7 ;
435/29; 435/325 |
Current CPC
Class: |
A61F 2002/4445 20130101;
A61L 27/3856 20130101; A61L 2430/38 20130101; A61F 2002/445
20130101; A61L 27/3817 20130101; A61L 27/3658 20130101; A61L
27/3612 20130101 |
Class at
Publication: |
424/93.7 ;
435/325; 435/29 |
International
Class: |
A61K 35/12 20060101
A61K035/12; C12Q 1/02 20060101 C12Q001/02; C12N 5/071 20100101
C12N005/071 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 12, 2003 |
DE |
103 59 830.8 |
Sep 6, 2004 |
DE |
10 2004 043 449.2 |
Claims
1. Use of degenerate prolapsed intervertebral disk tissue for the
production of a therapeutic agent for the treatment of
intervertebral disk defects.
2. The use according to claim 1, characterized in that during
growth, the intervertebral disk cells isolated from said degenerate
prolapsed intervertebral disk tissue are cultured in a cell culture
medium including 1-20% of added autologous serum, wherein the ratio
of alpha-MEM medium and HAM-'F12 medium is between 2:1 and 1:2, at
36.8-37.degree. C. in air containing 5% carbon dioxide and having a
humidity of 85-95%.
3. The use according to claim 1, characterized in that the isolated
intervertebral disk cells, following growth thereof in monolayer,
are cultured in a cell culture medium including 1-20% of added
autologous serum, wherein the ratio of alpha-MEM medium and HAM-F12
medium is between 2:1 and 1:2, at 36.8-37.degree. C. in air
containing 5% carbon dioxide and having a humidity of 85-95%,
thereby becoming capable of differentiating, forming matrix
structures comprising specific intervertebral disk matrix
proteins.
4. The use according to claim 1, characterized in that the isolated
intervertebral disk cells, following growth thereof in monolayer,
are frozen in a solution of 10% DMSO, 20% serum and 70% culture
medium, followed by thawing, so that their properties with respect
to the synthesis of specific matrix components and markers remain
unchanged and tissue structures consisting of intervertebral
disk-specific matrix proteins are formed in vitro and in vivo.
5. The use according to claim 1, characterized in that the cells
isolated from the intervertebral disk tissue are cultured in a
culture vessel with hydrophobic surface and tapering bottom,
thereby obtaining three-dimensional cell aggregates.
6. A method for the production of intervertebral disk cell
transplants, characterized in that intervertebral disk cells are
isolated from prolapsed degenerate intervertebral disk tissue
and/or affected intervertebral disk tissue and cultured as
three-dimensional aggregates with addition of autologous serum,
thereby obtaining three-dimensional intervertebral disk tissue
transplants.
7. An intervertebral disk tissue regeneration agent, which can be
obtained by isolating cells from degenerate intervertebral disk
tissue, followed by culturing, harvesting and using the cells as an
intervertebral disk regeneration agent.
8. The agent according to claim 7, characterized in that multiple
tissues are fused with each other.
9. The agent according to claim 1, characterized in that the agent
is a mixture of cultured cells and said three-dimensional
tissue.
10. The agent according to claim 1, which agent can be obtained by
providing the intervertebral disk cell transplants in a vessel with
tapering bottom and the intervertebral disk tissue aggregates in a
syringe for transplantation and transplanting them into the
intervertebral disk to be treated, namely, on the side opposite to
that of the first surgery of the intervertebral disk, by means of
injection using a puncture needle with slanted opening.
11. Use of the agents according to claim 1 in testing active
substances.
12. A cell-therapeutic formulation, comprising intervertebral disk
regeneration agents in accordance with claim 1.
Description
[0001] This application is a divisional of U.S. patent application
Ser. No. 10/596,357, filed Jun. 9, 2006, which is a National Phase
under .sctn.371 of PCT/DE2004/002761, filed Dec. 13, 2004, which
claims benefit of U.S. Provisional Patent Application 60/544,315,
filed Feb. 17, 2004, the disclosures of which are incorporated by
reference in their entirety.
[0002] The invention relates to a method for the in vitro
production of intervertebral disk cartilage cell transplants from
affected intervertebral disk tissue from patients and to the use
thereof as transplantation material for the treatment of affected
intervertebral disks. The invention also relates to a
three-dimensional, vital, and mechanically stable intervertebral
disk cartilage tissue and to the use thereof as transplantation
material for the treatment of affected intervertebral disks and in
testing active substances. Furthermore, the invention is directed
to the intervertebral disk cell transplants produced, the
transplantation technology, and the intervertebral disk cartilage
tissues produced, and to therapeutic formulations, e.g. injection
solutions containing said tissue and cell transplants.
[0003] Degeneration of intervertebral disks is initiated during
ageing or as a result of traumas, inducing acute and chronic pain
and instabilities in the vertebral column. More than 300,000
patients in Europe are suffering from a discopathy. Approximately
70% of the patients suffering from a prolapsed intervertebral disk
and being treated by means of discectomy are still suffering from
pain in their backs. In 10% of these patients, persistent severe
pain necessitates further surgical treatment (Yorimitsu et al.,
2001). This is due to a reduced height of the intervertebral disk
as a result of surgery, an increase of local strain on the
intervertebral disk tissue associated therewith (Brinckmann and
Grootenboer, 1991), and, in particular, lack of healing and
regeneration of destroyed and removed intervertebral disk tissue
(Lundon and Bolton, 2001; Meakin et al., 2001). In the course of
time, the instability of the affected intervertebral disk results
in degenerative changes of adjoining intervertebral disks, thereby
necessitating further surgical intervention and, in the worst case,
fusion of vertebral bodies or insertion of a prosthesis. Therefore,
biological repair or regeneration of intervertebral disks
represents the future in treating degenerate intervertebral
disks.
[0004] A well-known method for the biological regeneration of
tissue is cartilage cell transplantation using autologous cells,
which method has been used in the treatment of joint cartilage
defects. This method utilizes the potential of joint cartilage
cells of building up new tissue in vivo after transplanting the
cells. To this end, a joint cartilage biopsy is taken from the
patient, cartilage cells are isolated therefrom, grown by means of
cell culturing, and transplanted into the patient in the area of
the tissue defect e.g. by injection. There, they form new tissue,
thus completely filling up the defect. This method achieves
formation of tissue in the body after application of a cell
transplant. In principle, this methodology cannot be used in the
treatment of intervertebral disk degeneration because extraction of
starting material from an intact adjacent intervertebral disk of a
patient is not possible for ethical reasons and affected tissue
cannot be used a priori.
[0005] Initial approaches to biological replacement of
intervertebral disks have been using healthy intervertebral disk
tissue as starting material. Thus, Handley (U.S. Pat. No.
6,080,579) and Ferree (U.S. Pat. No. 6,340,369 B1) describe the use
of normal intervertebral disk tissue to isolate intervertebral disk
cells and combine these cells with a bioabsorbable carrier.
Similarly, a great deal of scientific work is based on the use of
normal intervertebral disk tissue: Okuma et al., 2000, Gruber et
al., 2000, Chelberg et al., 1995. However, a healthy intervertebral
disk of a patient cannot be used as a source of tissue to treat
another intervertebral disk because removal of tissue results in
destruction, degeneration and, as a consequence, loss of function
of this intervertebral disk.
[0006] Another approach involves the use of degenerate nucleus
pulposus tissue which is removed from the inside of a degenerate
intervertebral disk and worked up. This surgical intervention
further destroys the intervertebral disk which is degenerated and
destroyed anyway. On the one hand, work-up of the tissue is
suggested to be dehydration (U.S. Pat. No. 6,648,918) or
combination of cells present therein with a carrier material (U.S.
Pat. No. 6,569,442; US 2001/0020476 A1) and subsequent
re-transplantation into the degenerate intervertebral disks.
However, the dehydrated tissue does not include any living cells
and consequently does not represent a method of biological
re-generation. The suggested combination of nucleus cells or other
cells with a carrier material neither represents a purely
biological method and calls for the use of a suitable carrier
material which, for example, must be suitable in biomechanical
terms, which is to be formed and is degraded in the same way as new
tissue, which must not impair the formation of new tissue or, for
example, trigger immunologic reactions due to synthetic, allogenic
or xenogenic materials used in any event.
[0007] Another option of treatment involves the use of
intervertebral disks from other patients, which would therefore be
an allogenic transplantation (U.S. Pat. No. 6,344,058; Keith D K et
al., 2003). In this event, however, immunologic reactions represent
a problem, and mere introduction of a donor intervertebral disk
would probably not induce any biological regeneration of the
affected intervertebral disk. As a consequence of the problems
mentioned above, the general targets in treating degenerate
intervertebral disks and regenerating intervertebral disks are
therefore as follows: using medically and ethically justifiable
starting tissue or cell samples, avoiding destruction of other
intervertebral disks or of the affected, diseased intervertebral
disk of a patient, using materials from the patient only
(autologous therapy), finding optimum conditions of cell isolation
and culturing for the growth of intervertebral disk cells and
subsequent formation of intervertebral disk matrix, doing without
carrier materials so as to avoid immune reactions. The above
problems can be solved by transplanting a specific autologous cell
transplant or by transplanting a three-dimensional intervertebral
disk cartilage tissue which has been prefabricated outside the
body.
[0008] The object of the present invention was therefore to provide
methods for the production of intervertebral disk cartilage cell
transplants and stable vital intervertebral disk cartilage tissue
suitable for autologous transplantation, rapid re-development and
conservation of the intervertebral disk function. It is essential
that the starting material for the production of the cell
transplants can be collected in a medically and ethically
justifiable manner, and that the intervertebral disk cells cultured
in an autologous fashion do not undergo changes over the time
period from collection to transplantation and have high
proliferation and differentiation capacities.
[0009] Surprisingly, it was possible to demonstrate that affected
intervertebral disk tissue can be used as starting material. Up to
now, it has been assumed that degenerate tissue could not be used
to isolate adult cells in sufficient number, which cells would be
vital, have sufficient proliferation, and subsequently be even
capable of differentiating in a tissue-specific manner to form
intervertebral disk tissue, because tissue-specific cells in
tissues subject to degeneration change their properties with
respect to matrix synthesis, being destroyed and even replaced by
other cells with different properties lacking tissue
specificity.
[0010] Surprisingly, however, it was possible to isolate a
sufficient number of vital cells particularly from prolapsed
degenerate intervertebral disk tissue. Such prolapsed degenerate
intervertebral disk tissue consists of intervertebral disk portions
of the fibrous ring and of the pulpy nucleus, and the cells
isolated from the above two tissue areas (anulus fibrosus cells and
nucleus pulposus cells) even proliferate and differentiate
tissue-specifically in a particularly specific manner under the
autologous culturing conditions that are present. Hence, the above
mixed-cell transplants are suitable for a cell-based therapy to
restore the function of an intervertebral disk.
[0011] Thus, a method is described for the first time which allows
production of specific autologous mixed-cell intervertebral disk
transplants which, following transplantation in a damaged/affected
intervertebral disk, can save the intervertebral disk by forming
new intervertebral disk tissue and, as a consequence, restore the
neurological and mechanical functions of the vertebral column in
cases of discopathy or prolapsed intervertebral disk.
[0012] Even in advanced degeneration of an intervertebral disk,
i.e., degeneration or traumatic lesion of the outer layer of the
intervertebral disk (anulus fibrosus), the present invention
permits restoration and conservation of the neurological,
biological and mechanical functions of the intervertebral disk by
isolating mixed tissue cells from prolapsed, degenerate
intervertebral disks, which cells are subsequently cultured to form
autologous three-dimensional tissue without using carrier
materials. The isolated intervertebral disk tissue, especially the
intervertebral disk cells, are grown in a cell culture medium
preferably under autologous culturing conditions, adding serum from
the patient only. During growth, the intervertebral disk cells
isolated from degenerate, prolapsed intervertebral disk tissue are
preferably cultured in a cell culture medium including 1-20% of
added autologous serum, wherein the ratio of alpha-MEM medium and
HAM-F12 medium is between 2:1 and 1:2, at 36.8-37 .degree. C. in
air containing 5% carbon dioxide and having a humidity of 85-95%,
the synthesis of matrix and marker proteins by said cells remaining
unchanged.
[0013] In a likewise preferred fashion the isolated intervertebral
disk cells, following growth thereof in monolayer, are cultured in
a cell culture medium including 1-20% of added autologous serum,
wherein the ratio of alpha-MEM medium and HAM-F12 medium is between
2:1 and 1:2, at 36.8-37 .degree. C. in air containing 5% carbon
dioxide and having a humidity of 85-95%, thereby becoming capable
of differentiating, forming matrix structures comprising specific
intervertebral disk matrix proteins.
[0014] In a likewise preferred fashion the isolated intervertebral
disk cells, following growth thereof in monolayer, are frozen in a
solution of 10% DMSO, 20% serum and 70% culture medium, followed by
thawing, so that their properties with respect to the synthesis of
specific matrix components and markers remain unchanged and tissue
structures consisting of intervertebral disk-specific matrix
proteins are formed in vitro and in vivo.
[0015] The above-described features of unchanged synthesis of
marker and matrix proteins do not represent an object or desired
function to be achieved, but rather a consequence of the culturing
steps. The above features are mentioned merely for reasons of
clarification. Accordingly, the disclosure of these features is
intended to illuminate the consequences of the inventive steps of
use or processing.
[0016] Therefore, the formation of matrix structures comprising
specific intervertebral disk matrix proteins is not a preferred
property; rather, the formation of the above-mentioned matrix
structures is a result of the culturing conditions.
[0017] In a preferred fashion the cells isolated from
intervertebral disk tissue are cultured in a culture vessel with
hydrophobic surface and tapering bottom, thereby obtaining
three-dimensional cell aggregates.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 shows the histology of in vitro-produced
three-dimensional intervertebral disk cartilage tissues in the
cross-section of a microscope image. Vital differentiated cells
with extracellular matrix (M) are surrounded by a peripheral
proliferation zone P.
[0019] FIG. 2 shows the proliferation and high proliferation
capacity of intervertebral disk cells in mixed culture (anulus
fibrosus and nucleus pulposus) in the monolayer passage 2 (P2). The
number of cells per surface area over time is indicated in the
graph.
[0020] FIG. 3 shows the expression of disk-specific matrix proteins
and marker proteins by intervertebral disk cartilage mixed cells
following growth in monolayer culture and subsequent culturing
under three-dimensional cell culturing conditions. Expression of
components of native intervertebral disk cartilage in vivo
representing the most important structural proteins crucial for
intervertebral disk cartilage function are shown, including matrix
and regulatory proteins (aggregan, hyaline-specific proteoglycans,
and type I, II and III collagens). Expression of aggregan after 4
weeks is shown in 3a. Expression of hyaline-specific proteoglycans
detected by means of Safranin O staining after 4 weeks is shown in
3b. Expression of type I collagen after 2 weeks is shown in 3c.
Expression of type II collagen after 4 weeks is shown in 3d.
Expression of type III collagen after 4 weeks is shown in 3e.
[0021] FIG. 4 shows the fusing of five three-dimensional
intervertebral disk cartilage tissues during continued culture to
achieve larger transplants. The tissue spheres adhere to each other
and coalesce as shown, to undergo complete fusion after prolonged
culture.
[0022] FIG. 5 shows the expression of various matrix and regulative
proteins by disc derived chondrocytes cultured in monolayer for
different passages of cell culture [monolayer passage 2 (P2) and
monolayer passage 6 (P6)] and after freezing and thawing,
indicating that the formation of matrix and regulative proteins can
be retained.
[0023] In treatment using the intervertebral disk cell transplants
according to the invention, the outer envelope of the
intervertebral disk, i.e., the anulus fibrosus which has been
damaged by effusion of intervertebral disk tissue, advantageously
heals--prior to transplantation--in such a way that liquids, e.g.
the cell transplants produced, could not effuse from the interior
of the intervertebral disk. This time period is patient-dependent.
The intervertebral disk cell transplants are produced during this
period of time, and while growing in the cell culture, the
intervertebral disk mixed-cells receive their tissue-specific
properties with respect to their differentiation potential and, as
a consequence, the success of transplantation. In contrast, this
potential is reduced when separately culturing the anulus fibrosus
and nucleus pulposus cells, and some intervertebral disk-specific
markers will not be expressed following transfer into the
three-dimensional environment. For this reason, it is only mixed
cultures that are particularly suitable in building up
intervertebral disk tissue after transplantation in a degenerate
intervertebral disk.
[0024] In addition, the cell and tissue transplants produced in
vitro should not induce any immunologic reactions in the organism
containing the transplant. Surprisingly, it was found that the
cells and tissues produced in an autologous manner according to the
invention do not induce any immunologic reactions.
[0025] The affected intervertebral disk tissue collected from the
patient can be further processed in various ways: (a) The
intervertebral disk mixed cells are isolated from the biopsies
according to conventional methods, using enzymatic digestion of the
tissue, migration, or reagents recognizing the target cells. Adding
autologous serum only, without addition of exogenic
growth-promoting compounds and without addition of antibiotics, the
above cells are cultured in cell culture vessels in conventional
culture medium until a sufficient amount of cells is available
(FIG. 2). This period of time is made as short as possible so as to
avoid changes in their phenotypic properties. After sufficient cell
propagation, the cells are harvested, and the cell transplant
consisting of an intervertebral disk cell suspension is ready for
therapeutic applications.
[0026] According to the invention, the isolated intervertebral disk
cells are used in mixture, i.e., the cells are not separated
according to anulus fibrosus and nucleus pulposus cells--tissue
portions of which are included in the prolapsed intervertebral disk
tissue--and cultured separately or as single-type cells. It is
precisely these mixture conditions that later achieve improved
intervertebral disk-specific differentiation of the cells (see FIG.
3). Hence, the above autologous mixed culturing technique for
growing intervertebral disk cells--following transfer thereof into
a three-dimensional environment--allows intervertebral
disk-specific differentiation of the thus-cultured cells for the
first time. (b) In another method the isolated intervertebral disk
cells are pre-cultured and briefly grown without passaging.
Thereafter, the pre-cultured cells are harvested, frozen, and
stored in deep-frozen state until used in transplantation. Prior to
transplantation, the cells are thawed and cultured further together
with autologous serum in conventional cell culture medium until a
sufficient number of cells is reached. Following sufficient
propagation of the cells, the cells are harvested, thus providing
the cell transplant consisting of an intervertebral disk cell
suspension. Surprisingly, it was noted that the intervertebral disk
cells do not lose their specific properties with respect to the
synthesis of specific marker and matrix proteins as a result of
such freezing and thawing (see FIG. 5). (c) Another preferred
procedure likewise utilizes patient-derived affected intervertebral
disk tissue as starting material. The tissue-building cells are
isolated from the biopsies from anulus fibrosus and nucleus
pulposus according to conventional methods, using enzymatic
digestion of the tissue, migration, or reagents recognizing the
target cells. Thereafter, these mixed cells are initially cultured
in monolayer under autologous conditions, using a standard culture
medium, until a sufficient number of cells is reached, subsequently
transferred into cell culture vessels with hydrophobic surface and
tapering bottom and cultured in suspension therein until a
three-dimensional cell aggregate is formed which includes at least
40% by volume, preferably at least 60% by volume, and up to a
maximum of 99% by volume of de novo-synthesized extracellular
matrix (ECM) having differentiated cells embedded therein. By
taking small samples, these values can be determined by a person
skilled in the art. The cell aggregate having formed has an outer
region wherein cells capable of proliferation and migration are
present (see FIG. 1).
[0027] According to the invention, the isolated intervertebral disk
cells are used in mixture, i.e., the cells are not separated
according to anulus fibrosus and nucleus pulposus cells and
cultured separately or as single-type cells. It is precisely under
such mixture conditions that intervertebral disk-specific
differentiation of the mixed cells is promoted in the
three-dimensional culture and, as a consequence, in the formation
of three-dimensional intervertebral disk tissue, and the expression
of intervertebral disk-specific markers can only be achieved in
such cultures, with formation of three-dimensional intervertebral
disk tissue being promoted when using said mixed cultures (see FIG.
3). Hence, the above autologous mixed culturing technique permits
production and use of an intervertebral disk-specific tissue
transplant for the first time, which transplant is produced in an
autologous fashion from degenerate, prolapsed intervertebral disk
tissue.
[0028] The intervertebral disk mixed cells isolated from affected
intervertebral disk tissue and used to produce autologous
three-dimensional intervertebral disk cell aggregates such that the
collected cells are integrated in a de novo-synthesized tissue
survive even after a prolonged time of culturing, i.e., the cells
inside the aggregates do not die. With increasing culturing time,
the cells inside the aggregates undergo differentiation to form
intervertebral disk cartilage tissue consisting of ECM,
differentiated cells and a peripheral proliferation zone (see FIG.
1). During differentiation in the autologous cell culture, the
spacing between the aggregated cells steadily increases due to
formation of tissue-specific matrix (see FIG. 3, comparison of (3c)
with (3d)). A tissue histology develops inside the intervertebral
disk tissue produced in vitro, which is highly similar to natural
tissue. During the further course of production of intervertebral
disk cartilage tissue, a "proliferation zone" is formed at the
boundary thereof. This zone is invaluably advantageous in that,
following incorporation of the thus-formed intervertebral disk
cartilage tissue in degenerate intervertebral disks, the cells
situated in this peripheral zone are capable of migrating to make
active contact with the surrounding tissue and/or enable
integration of the in vitro produced intervertebral disk cartilage
tissue in the environment thereof. Thus, the tissue-specific cell
aggregates produced are excellently suited for use in the treatment
of degenerate intervertebral disks and in in vivo neogenesis of
intervertebral disk tissue.
[0029] In view of the biomechanical strain on the intervertebral
disks immediately after treatment, and with respect to the target
of restoring the intervertebral disk in its height at the same time
with transplanting the intervertebral disk cartilage tissue, it can
be advantageous to transplant larger pieces of tissue at an early
stage. In this event, at least two, or preferably more of the
intervertebral disk cartilage tissues obtained in vitro are fused
by continuing culturing thereof under the same conditions and in
the same culture vessels as described above until the desired size
is reached (see also FIG. 4).
[0030] A medium usual for both suspension and monolayer culture,
e.g. Dulbecco's MEM, with addition of serum, can be used as cell
culture medium. It is preferred to use DMEM and HAMS at a ratio of
1:1. However, to avoid immunologic response in the patient to the
tissue produced in vitro, it is preferred to use autologous serum
from the patient as serum. It is also possible to use xenogenic or
allogenic serum.
[0031] According to the invention, no antibiotic, fungistatic
agents or other auxiliary substances are added to the culture
medium. It has been found that only autologous or allogenic
culturing of the cells and cell aggregates and culturing with no
antibiotic and fungistatic agents allows non-affected proliferation
and differentiation of the cells in the monolayer culture and
undisturbed formation of the specific matrix within the cell
aggregates. Furthermore, by avoiding additives during the
production, any immunologic reaction is excluded when incorporating
the tissue produced in vitro in a human or animal organism.
[0032] The size of the intervertebral disk tissue produced depends
on the number of incorporated cells per volume of culture medium.
For example, when incorporating 1.times.10.sup.7 cells in 300 .mu.l
of culture medium, three-dimensional intervertebral disk cell
aggregates about 500-700 .mu.m in diameter are formed within one
week. Another option would be in vitro fusion of small cell
aggregates to form larger ones--as described above--and
incorporation of the latter in the intervertebral disk. According
to the invention, it is preferred to use between 1.times.10.sup.4
and 1.times.10.sup.7 cells in 300 .mu.l of culture medium to
produce small cell aggregates, more preferably 1.times.10.sup.5
cells. Depending on the cell type and patient-specific
characteristics, the intervertebral disk tissues having formed in
vitro after several days are then cultured in a suitable culture
medium for at least 2-4 weeks to induce formation of the
tissue-specific matrix. From about one week of culturing on, it is
possible in special cases to fuse individual in vitro
intervertebral disk tissues so as to increase the size of the
tissue patch.
[0033] As cell culture vessels for the inventive culturing in
suspension, those having a hydrophobic, i.e., adhesion-preventing
surface, such as polystyrene or Teflon, can preferably be used.
Cell culture vessels with a non-hydrophobic surface can be
hydrophobized by coating with agar or agarose. Further additives
are not required. Preferably, well plates are used as cell culture
vessels. For example, 96-well plates can be used to produce small
cell aggregates, and 24-well plates to produce said fused
aggregates.
[0034] The invention is also directed to a surgical technique of
transplanting the intervertebral disk cells and the in
vitro-produced three-dimensional intervertebral disk cartilage
tissues into a damaged intervertebral disk. According to the
invention, transplantation is effected by injecting the
intervertebral disk cells under fluoroscopic control, following
disinfection of the skin, sterile covering of the skin area and
local anesthesia, and--in a preferred fashion--strictly avoiding
the use of contrast media.
[0035] To this end, the intervertebral disk cell
transplants--following production in a laboratory--are filled in
special transportation tubes with tapering bottom, rounded or
sharp, and--in the operating room--drawn up in a syringe through a
puncture needle with e.g. a slant-ended cannula. Complete uptake of
the solution containing the cells is made possible especially by
the tapering bottom of the transportation vessel and the
slant-ended cannula. To ensure delivery of the cells in the
intervertebral disk without damaging the cells and with lowest
possible loss of liquid, i.e. cells, the puncture needle
essentially has an inner diameter of 0.4 to 2 mm. According to the
present invention, transplantation by injecting the intervertebral
disk cells in the intervertebral disk to be treated is effected
particularly at the side opposite to the previously operated side
of the intervertebral disk (removal of intervertebral disk
prolapse), using a slant-ended puncture needle. According to the
invention, injection of the cell transplant takes place under
fluoroscopic control because the actual delivery of cells into the
intervertebral disk interior must be monitored. Conventionally, the
use of contrast media is possible which, however, damage the cells,
thereby preventing successful cell transplantation. Following
delivery of the intervertebral disk cells in the intervertebral
disk space, the puncture needle is retracted from the
intervertebral disk. Preferably, the patient then is ordered to
strict bed rest for 12 hours, followed by 12-24 hours of regular
bed rest and 24-48 hours bed rest with physiotherapeutic exercises.
Thereafter, the vertebral column is stabilized e.g. for some weeks
using a conventional suitable orthesis.
[0036] As in the case of intervertebral disk cells, transplantation
of the three-dimensional in vitro-produced intervertebral disk
cartilage transplants is effected by means of a puncture needle, in
particular. To make sure that mechanical and therefore biological
damage of the intervertebral disk cartilage transplants is avoided
during injection, a puncture needle with a diameter of at least 500
.mu.m is used. According to the invention, essentially single
passage through the puncture needle is effected to avoid mechanical
damage of the intervertebral disk cartilage transplants. For this
reason, the intervertebral disk cartilage transplants are
transferred in a syringe following production thereof in a
laboratory, on which syringe the puncture needle merely has to be
placed in the operating room. According to the invention, the
puncture needle used in this case must also have a slanted opening
so as to allow rapid delivery--by virtue of the enlarged area of
delivery--of the intervertebral disk cartilage transplants in a
smallest possible liquid volume (delivery volume). Thereafter,
injection is effected as described for the cell transplants
above.
[0037] The invention is also directed to therapeutic formulations
comprising the intervertebral disk cell transplants and
intervertebral disk cartilage tissue of the invention, e.g.
injection solutions.
[0038] The invention is also directed to the use of the
intervertebral disk cartilage tissues of the invention in testing
active substances which e.g. have influence on the formation and
differentiation of matrix and cells. To this end, the
intervertebral disk cell aggregates are produced in accordance with
the invention, medications to be tested are added at varying stages
of maturing, and a variety of parameters of in vitro intervertebral
disk tissue production and maturing are characterized. Compared to
conventional testing of drugs in animals or tumor cell systems,
these tests are patient-specific owing to the use of autologous
material only and allow individual diagnosis.
[0039] Without intending to be limiting, the invention will be
illustrated in more detail with reference to the examples.
EXAMPLES
Example 1
[0040] Production of Intervertebral Disk Cell Transplants
[0041] Intervertebral disk cartilage cells from anulus fibrosus and
nucleus pulposus are isolated from affected intervertebral disk
tissue, using enzymatic digestion by incubation with collagenase
solution. Following separation of the isolated cells from
undigested cartilage tissue, the cells in the form of a mixed-cell
population are transferred in cell culture flasks and, following
addition of DMEM/HAMS F12 culture medium (1/1) and 10% autologous
serum from the patient, incubated at 37 .degree. C. and 5%
CO.sub.2. The medium is exchanged twice a week. After reaching the
confluent stage, the cell layer is washed with physiological saline
solution and harvested from the cell culture surface using trypsin.
After another wash, the intervertebral disk cells are transferred
in physiological saline solution and provided for
transplantation.
[0042] The differentiation potential of the intervertebral disk
cells included in the cell transplant was demonstrated in an in
vitro model. Intervertebral disk-specific matrix proteins and
marker proteins are expressed (FIG. 3) and a intervertebral
disk-specific tissue formed in this way.
Example 2
[0043] Transplantation of Intervertebral Disk Cartilage Cells
[0044] The intervertebral disk cell transplants produced in Example
1 (minimum: 1,000 cells, maximum: 100 million cells), preferably
about 1 million intervertebral disk cartilage cells, were taken up
in physiological saline solution and injected in an affected
intervertebral disk. It was noted, among other things, that the
water content was rising and the height of the intervertebral space
was maintained in the treated intervertebral disk, both of the
above being due to the matrix proteins synthesized by the
intervertebral disk cartilage cells.
[0045] The in vitro-produced intervertebral disk cell transplants
of the invention are accepted by patients, ensure rapid integration
of the cells capable of proliferation and migration, as well as
regeneration of the intervertebral disk tissue as a result of the
differentiating ability of the cells included therein. Therefore,
the intervertebral disk cell transplants allow rapid re-development
of intervertebral disk tissue, rapid convalescence of patients, and
restoration of the intervertebral disk function.
Example 3
[0046] In vitro Production of Intervertebral Disk Cartilage
Tissue
[0047] Intervertebral disk cartilage cells from anulus fibrosus and
nucleus pulposus are isolated from prolapsed intervertebral disk
tissue, using enzymatic digestion by incubation with collagenase
solution. Following separation of the isolated cells from
undigested tissue, the cells in the form of a mixed culture are
transferred in cell culture flasks and, following addition of
DMEM/HAMS F12 culture medium (1/1) and 10% autologous serum from
the patient, incubated at 37 .degree. C. and 5% CO.sub.2. The
medium is exchanged twice a week. After reaching the confluent
stage, the cell layer is washed with physiological saline solution
and harvested from the cell culture surface using trypsin. After
another wash, 1.times.10.sup.5 cells each time are transferred in a
cell culture vessel coated with agarose. After one day, the first
cells have arranged into aggregates. These aggregates are supplied
with fresh medium at regular intervals and cultured for at least 2
weeks.
[0048] The structure of the resulting intervertebral disk cartilage
tissue is represented in the microscopic image in FIG. 1 which
shows the cross-section of an intervertebral disk tissue produced
according to the invention, with ECM as a zone of reduced
proliferation and formation of tissue-specific matrix proteins and
P as outer proliferation zone.
[0049] Expression and deposition of intervertebral disk-specific
matrix components and regulatory proteins such as aggregan (FIG.
3a), hyaline-specific proteoglycans (FIG. 3b), collagen type I
(FIG. 3c), collagen type II (FIG. 3d) and collagen type III (FIG.
3e) was detected in the above in vitro intervertebral disk tissues.
These are components of native intervertebral disk cartilage tissue
in vivo, representing the most important structural proteins which
are of crucial importance for the function of intervertebral disk
cartilage.
[0050] Surprisingly, when cultured as a mixture of anulus fibrosus
and nucleus pulposus, the intervertebral disk cells isolated from
affected intervertebral disk tissue show high proliferation
capacity (FIG. 2) and very high differentiation potential for the
formation of intervertebral disk-specific matrix proteins and
regulatory proteins (see also FIG. 3), and their properties can be
retained by the procedure of freezing and thawing (see also FIG.
5).
Example 4
[0051] Transplantation of Intervertebral Disk Cartilage Tissue
[0052] The intervertebral disk cartilage tissue produced in Example
3 (about 10 to 1000 tissue patches, each comprising
1.times.10.sup.5 cells), preferably 100 tissue patches, in
physiological saline solution was taken up in a syringe in the
laboratory and injected into the intervertebral space of affected
or damaged intervertebral disks using a puncture needle with
slanted opening. The intervertebral disk cartilage tissue produced
in vitro according to the invention is well-accepted by patients
and, in addition to realizing the mechanical function of the tissue
produced, ensures rapid integration of the resulting tissue patch
in the outer layers of the aggregate by the cells capable of
proliferation and migration, as well as regeneration of the
intervertebral disk tissue as a result of the differentiating
ability of the cells included therein. Hence, structure and
function of the tissue patches allow rapid re-development of
intervertebral disk tissue, rapid convalescence of patients, and
restoration of the intervertebral disk function.
[0053] FIG. 4 shows five intervertebral disk tissues undergoing
fusion. As can be seen, the tissue spheres adhere to each other,
and coalesce, so to speak, the boundary between any two
intervertebral disk tissues is no longer recognizable. After a
prolonged period of culturing, the intervertebral disk tissues
undergo complete fusion to form a larger in vitro tissue patch. The
structure of the larger cell aggregates thus obtained is comparable
with in vitro intervertebral disk tissue. They can include up to
maximum of 99% ECM, and the resulting cells are vital.
* * * * *