Note: Descriptions are shown in the official language in which they were submitted.
r
_.
Hemocompatible surfaces and process for the production thereof
The present invention concerns hemocompatible surfaces which
are characterized in that constituents of the outer layers of
blood cells and/or mesothelial cells are applied and/or
incorporated onto and/or into the surfaces of materials.
The present invention further concerns a process for
manufacturing hemocompatible surfaces~and their use in
extensive fields of health, in medicine, dentistry, surgery,
cosmetics and/or fields having direct contact with blood,
tissue and/or other body fluids.
In the case of vertebrates, blood coagulation is a complex
process which temporarily protects against critical losses of
blood in the case of injury. The blood coagulation system is
activated, among other things, by contact with unphysiologic,
i. e., 'exogenous' substances in this case. Substances which
actively suppress the blood coagulation system are also
referred to as anti-thrombogenic. Substances which do not even
activate the blood coagulation system are defined as non-
thrombogenic.
Especially in the case of invasive operations, the activation
of the blood coagulation system is a serious problem for the
patient. This is, in particular, the case for people dependent
on implants, such as intra-coronary stents, cardiac valves,
prosthetic devices, artificial vascular systems, dialysers, or
oxygenators, catheters, biosensors etc.. Contact with
surgical suture materials can also cause problems.
Until now, in order to prevent the formation of critical
occlusions of vessels (thrombi), the blood coagulation system
CA 02363119 2001-08-24
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2
has been deactivated or actively suppressed. This is normally
done by the administration of anti-thrombogenic medicine, so-
called anticoagulants, which however, have many serious side
effects for the patient, such as thrombocytopenia, nausea,
vomiting, hair loss, haemorrhagic skin gangrenes, higher
tendency to bleed etc.. Moreover, if intra-coronary stents
or cardiac valves are used, even the complete medicamentous
suppression of blood coagulation often does not sufficiently
prevent the formation of thrombosis, which can cause death.
In extensive fields of health, in medicine, dentistry,
surgery, cosmetics or, in general, fields having contact with
blood and/or other body fluids in invasive operations, it is
therefore very important to avoid the above-mentioned serious
side effects caused by anticoagulants.
From prior art, various processes are known which are intended
to make unphysiologic ~foreig~ surfaces' more blood compatible
(hemocompatible) or histocompatible by coating with different
substances.
DE 28 31 360, for example, describes a process for coating a
surface of a medical article with a substance (heparin) which
actively suppresses the coagulation system, i. e., is anti-
thrombogenic. Said substance, however, has the disadvantage of
serious side effects for the patient, as already mentioned
before by way of example.
In DE 44 35 652, materials are coated with a thin coat of
lacquer of polymers into which medicinal agents can be
additionally incorporated, wherein said coat of lacquer
permanently degrades in the body and is thus released. The
disadvantages of this method are, first of all, that because
of the permanent degradation of the coating only a temporally
limited effect can be achieved. Secondly, due to the permanent
f
3
separation of particles of lacquer, there is__a,high danger of
the formation of thrombosis, which can cause embolisms.
DE 196 30 879 exclusively uses chemically modified derivatives
of polysaccharides for coating substrates. There are various
disadvantages regarding this process, ranging from excessive
preparative expenses to synthesis steps including many stages,
a wide range of undesirable side reactions and poor
exploitation up to worse properties of the derivatives in
every respect when compared to commercially available anti-
thrombogenic substances such as heparin. _
Verhagen et al. (British Journal of Heamatology, 1996, 95:
542-549) describes the use of entire living cells of the
endothelium or the mesothelium for the colonisation of
implants. The disadvantage of using entire cells is the fact
that, due to specific cell surface proteins, immune reactions
are caused, which cause rejection reactions against the coated
implants for the patients. Substances inducing such an immune
reaction are also called immunogenic. To prevent a rejection
by immune reactions, it is necessary that exclusively cell
material of the patients themselves is used in this process.
This is a further disadvantage because considerable time and
costs are involved in culturing these cells. A further problem
regarding the use of entire cells are the high shearing forces
to which these cells are subjected in the blood stream. This
leads to an increased degradation of the cells at the
surfaces, which has a negative effect on the durability of the
coated implants.
Also WO 93/01843, WO 95/29712 and DE 195 05 070 describe the
use of entire living endothelial cells for coating
unphysiologic materials or the use of substances contributing
to the growing of living endothelial cells on artificial
materials. But also in these cases, all processes are based on
CA 02363119 2001-08-24
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4
the cultivation of living endothelial cells, which involves
the disadvantages mentioned above with respect to the time
. required and the cost involved or the considerable limitation
that the coated material cannot be used universally, but has
to be produced separately for every patient.
From patent specification DE 36 39 561, the production of
substrates coated with the specific endothelial cell surface
proteopolysaccharide HS-I is known. The disadvantage of the
process is the fact that also in this case considerable
amounts of endothelial~cells of the patients-themselves are
required for isolating these components. This requires for
every patient a time-consuming and cost-intensive cultivation
of his endogenous endothelial cells, which, in addition, is
followed by a costly preparation of the proteopolysaccharide
HS-I. Therefore, the mass production of HS-I and thus an
economic use of this process for coating implants cannot be
realized.
Accordingly, the object of the present invention is to provide
blood compatible (hemocompatible) or histocompatible surfaces
which do not show the disadvantages mentioned above and are,
at the'same time, suitable for mass production.
According to the invention, the object is solved by means of
hemocompatible surfaces characterized in that they contain as
the materials artificial and/or natural organic and/or
inorganic compounds and/or mixtures thereof and/or materials
having contact with blood and/or other body fluids in invasive
operations and/or animal organs and/or organ parts, and
constituents of the outer layer of blood cells and/or
mesothelial cells are applied and/or incorporated onto and/or
into the surface of said materials.
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i I
The hemocompatible surfaces of the invention__thus
substantially imitate the outer surface of blood and/or
mesothelial cells, synonymous with the imitation of the
natural surface of non-thrombogenic cells and/or tissue.
The blood coagulation system is therefore neither activated
nor actively suppressed by the hemocompatible surfaces.
Accordingly, a blood coagulation which is, for example, caused
by secondary injuries (cuts or the like) can take place in a
completely natural and undisturbed way.
A further advantage of the present invention is the fact that
an adhesion of cells such as thrombocytes on the
hemocompatible surfaces according to the invention does not
occur. This is desired by the invention because the risk of
the formation of thrombi, i. e., the danger of a thrombosis
(embolism) for the patient treated is minimized thereby. The
hemocompatible surfaces according to the invention do no cause
any side effects.
According to the invention, the hemocompatible surfaces are
further characterized by the fact that they are non-
thrombogenic in the long term. This means that their
advantageous properties are not used up in the course of time,
which is, for example, the case for pharmaceutically active
systems (for example release system). For this reason, the
surfaces according to the invention are also suitable for
permanent use, so that additional burdening and risks for the
patients by repeated invasive operations for renewing the
implants are minimized.
According to the invention, the hemocompatible surfaces
contain as the materials artificial and/or natural organic
and/or inorganic compounds and/or mixtures thereof and/or
materials having contact with blood and/or other body fluids
in invasive operations and/or animal organs and/or organ
parts, and constituents of the outer layers of blood cells
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6
and/or mesothelial cells are applied and/or incorporated onto
and/or into the surfaces thereof.
In the sense of the invention, materials refer to any
materials which, according to the invention, are suitable for
being loaded with cell constituents. Also comprised are any
materials which can come into contact with blood and/or body
fluids during invasive operations or in connection with
respective postoperative care.
Organic compounds refer, for example, to synthetically
produced or naturally occurring high-molecular weight substances and
their derivatives. Examples for these are, among others, any
kinds of plastics, elastomers, silicones or fibrous
substances. They include, for example, polyethylenes (PE),
polyvinyl chlorides (PVC), polyurethanes (PUR), polyamides
(PA), phenoplasts (PF), aminoplasts, polystyrene, polyester,
resins, silicones, rubbers, man-made fibers, cellulose fibers,
cellulose membranes, protein fibers, collagens, as well as
derivatives thereof or combinations thereof. Further comprised
according to the invention are mixtures of these polymers, so-
called polymer blends.
In a special embodiment of the present invention, as
materials, the hemocompatible surfaces according to the
invention can include animal organs, organ parts or vascular
systems. They can, for example, be cardiac valves and/or
vascular systems, wherein pigs or cattle are especially
suitable as sources.
Examples for inorganic compounds included by the
hemocompatible surfaces according to the invention are metals,
metal oxides, alloys or ceramics, glasses and/or minerals as
well as derivatives thereof or any possible combinations
and/or mixtures thereof. According to the invention, any
CA 02363119 2001-09-21
possibilities of combination of materials are possible. The
examples explain the present invention in greater detail, but
are not intended to be limiting.
According to the invention, constituents of the outer layers
of blood cells and/or mesothelial cells are incorporated
and/or applied into and/or onto the surface of the materials.
In one embodiment of the present invention, the hemocompatible
surfaces include in and/or on the surface of materials
glycoproteins, preferably glycophorins. Said glycophorins
are, among other things, characterized by non-
thrombogenic properties, and are therefore excellently
suitable for making hemocompatible surfaces according to the
invention.
The glycophorins of the outer layer of the erythrocytes,
determine, among other things, the blood group to which a
human being belongs. Analogously to the different blood groups
A, B, AB and 0, the corresponding erythrocytes contain
glycophorin A, glycophorin B or glycophorin 0 or respective
mixtures thereof.
A possible immunological response by cross-reactions of blood
groups which are not compatible with each other, i. e.,
clotting of blood (coagulation) can be avoided in a simple way
by matching with respect to the blood group of the patient
treated and the glycophorins applied and/or incorporated onto
and/or into the surfaces of materials of the hemocompatible
surfaces of the invention which are intended for application,
wherein said matching is carried out before the invasive
operation. Respective blood tests are common practice in
laboratories and, accordingly, are carried out routinely.
Provided that the blood-group compatibility is observed,
hemocompatible surfaces containing glycophorin can thus also
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be used universally, i. e., they are not restricted to only
one patient.
The present invention further concerns hemocompatible surfaces
including on and/or in the surfaces of the materials
oligosaccharide, polysaccharide and/or lipid portions of the
glycoproteins, glycolipids and/or proteoglycans from the outer
layer of blood cells and/or mesothelial cells.
In a further embodiment of the present invention, the
hemocompatible surfaces contain glycosphingolipids on and/or
in the surfaces of the materials.
The hemocompatible surfaces of the present invention further
can contain as the oligosaccharide or polysaccharide portions
of the proteoglycans hyaluronic acids, chondroitin sulfates,
dermatan sulfates, heparan sulfates, keratan sulfates or
mixtures thereof. In a preferred embodiment of the present
invention, the hemocompatible surfaces contain heparan sulfate
of the erythrocyte plasma membrane of animal and/or human
origin.
The hemocompatible surfaces according to the invention do not
show any side effects, which are caused, for example by
chemically or pharmaceutically active coatings.
The above-mentioned constituents of the blood and/or
mesothelial cells are non-immunogenic cell constituents.
Accordingly, the hemocompatible surfaces according to the
invention are characterized in that they are also non-
immunogenic. This means they do not cause an immune reaction
for the patient, which minimizes the danger of rejection of
the hemocompatible surfaces.
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' 9 '
According to the invention, the hemocompatible,surfaces are
non-thrombogenic and/or non-immunogenic.
A further advantage is the fact that almost no degradation
takes place at the hemocompatible surfaces due to the firm
attachment of the non-thrombogenic constituents of the outer
layers of the blood and/or mesothelial cells on the materials
according to the invention. The danger of the formation of
embolisms by thrombosis is thus minimized. Furthermore, there
is no accumulation of cells such as thrombocytes on the
hemocompatible _surfaces according to the invention. This also
minimizes the danger of thrombosis.
The subject matter of the invention further comprises a
process for the production of the hemocompatible surfaces
according to the invention, wherein glycophorins,
oligosaccharide, polysaccharide and/or lipid portions of the
glycoproteins, glycolipids and/or proteoglycans from the~outer
layer of blood cells and/or mesothelial cells are isolated,
and these cell constituents are applied and/or incorporated
onto and/or into the surfaces of materials of artificial
and/or natural organic and/or inorganic compounds and/or
mixtures thereof and/or materials having contact with blood
and/or other body fluids in invasive operations and/or animal
organs and/or organ parts by physical or chemical bonding.
According to the invention, the constituents of the outer
layer of blood cells are isolated from whole blood and/or
from cell fractions obtained therefrom of human or animal
origin. This means that the cell constituents are isolated
from erythrocytes, leucocytes and/or thrombocytes or mixtures
thereof. Preferred are mixtures of erythrocytes and
leucocytes. Especially preferred are erythrocytes.
CA 02363119 2001-08-24
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The constituents of the outer layer of mesothelial cells are,
according to the invention, isolated from omentum, peritoneum
and/or inner organs.
A cheap and easily accessible source for these starting
materials can be waste from slaughtering, for example.
The isolation of the constituents of the outer layer of the
blood cells, mesothelial cells or of the tissue rich in
mesothelial cells is~carried out in a common manner in this
case. The following processes or combinations:thereof are, for
example, possible: comminution, extraction, filtration,
precipitation, gel filtration, ion exchange chromatography,
affinity chromatography, electrophoresis, enzymatic or
chemical degradations, drying, dissolution, dialysis,
ultrafiltration etc..
According to the invention, for applying and/or incorporating
the cell constituents onto and/or into the surface of the
materials, a chemical immobilization, photoimmobilization,
adhesion, drying process or a combination thereof is carried
out. Covalent, ionic, secondary valence or electrostatic or
adhesive bonds or combinations thereof can be formed between
the constituents of the outer layer of the cells and the
surfaces of the materials in this case. Preferably, the
application or integration of the constituents of the outer
cell layer onto/into the surfaces of materials is carried out
by covalent bonds.
A special advantage of the present invention is the fact that
the production process according to the invention combines
enormous economical advantages compared to the processes known
until now, and, accordingly, the hemocompatible surfaces
according to the invention are suitable for mass production.
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The reasons for this are, for example, that, according to the
invention, cell constituents and not living cells are used,
that no endogenous (endothelial) cells of the patient must be
used, that the starting material for the isolation of said
cell constituents is cheap and available in big amounts (waste
from slaughtering), so that cell cultivation, which is very
time-consuming and cost-intensive, is not necessary. A further
advantage of the hemocompatible surfaces according to the
invention is the fact that they can be used universally and
are not restricted to the use for only one patient. Above all,
in the case of emergency operations, this advantage is
essential for a patient's life.
There is a wide range of fields of applications in which the
present invention can be used. The present invention concerns
the use of hemocompatible surfaces in extensive fields of
health, in medicine, dentistry, surgery or cosmetics and/or in
fields having contact with blood, tissue and/or other body
fluids during invasive operations.
In the following, the invention is described i~ greater detail
with reference to the examples, which, however, are not
intended to be limiting:
1.) Isolation of erythrocyte plasma membrane heparan sulfate:
One liter of erythrocytes which have been washed free of serum
are suspended in 1 liter of a 0.154 molar phosphate buffer pH
7, and 1 U/ml papain is added. After 2 hours of incubation at
56~C, centrifuging takes place at 3000 g for 20 minutes, and,
subsequently, the supernatant is decanted. In this
supernatant, 100 ml of DEAF Sepharose~ CL-6B ion exchanger gel
of the company Pharmacia Biotech are suspended. The gel loaded
in this way is still washed three times in a 0.1 molar saline
CA 02363119 2005-09-29
' 12
solution and filled into a chromatographic column. The elution
takes place by means of a linear sodium chloride gradient in
the range of 0.1 to 0.8 moles/1 over an entire elution volume
of 2 liters. 200 fractions of a volume of 10 ml each are
collected. The fractions showing a positive color reaction
with dimethylmethylene blue (DN~IB) of the company Fluka
according to the method described by Chandrasekhar et al
(Analytical Biochemistry, 161 (1987): 130-108) are united. The
solution of the collected fractions is narrowed down at 26.7
hPa (20 torrs) and 40°C and dialysed against water. The
dialysate is set to a volume of 100 ml and a concentration of
0.03 moles/1 of sodium acetate, 0.073 moles/1 of
tris (tris(hydroxymethyl)aminomethane of the company Fluka)
and pH 8.0, 1 U of chondroitinase ABC is added, and incubation
takes place at 37°C for 15 hours. After dialysing against
water and narrowing down under water jet vacuum, the resulting
solution is again applied onto a column with 100 ml of DEAE
Sepharose CL-6B of the company Pharmacia Biotech. and eluted
as described before. The DN>MB positive gradient fractions are
dialysed, narrowed down under water jet vacuum to a volume of
1 ml and chromatographed on a column for preparative gel
filtration (60 cm x 2 cm) using a Sephacryl~ S-300 gel
(Pharmacia Biotech.). 60 fractions of a volume of 2 ml each
are collected, detected with DrM~iB, and the positive fractions
are united. After repeated dialysis and lyophilisation, the
purified erythrocyte plasma membrane heparan sulfate will be
obtained.
2.) Isolation of leucocyte surface proteo-chondroitin sulfate:
One liter of citrate blood is centrifuged for 10 minutes in a
centrifuge with a swing-out rotor at 3000 g, and the
supernatant plasma is drawn off. The cell sediment is mixed
with 2 liters of a 1% ammonium oxalate solution cooled to 4°C,
CA 02363119 2005-09-29
13
and is incubated for 30 minutes at the same temperature. After
minutes of centrifugation at 500 g, the red supernatant is
discarded and the pellet is suspended in 2 liters of a 1%
ammonium oxalate solution cooled to 4°C, centrifuged for 5
minutes at 500 g, and the washing process as described above
is repeated two more times. The supernatant which is now
colorless is discarded, and the washed cell sediment (yield:
12 x 10' - 10 x 109 cells in 2 liters of triton X-100 buffer
(0.5 % triton x-loo, to mM tris-HGI, 15o mM NaG, pH s) is
lysed for 2 hours at 25°C under constant stirring. The
detergent extract is centrifuged for 60 minutes at 10,000 g,
decanted, and in the supernatant, 10 ml of DEAE Sephadex~ A50
ion exchanger gel of the company Pharmacia Biotech are
suspended and sedimentated. The gel loaded in this way is
still washed three times in a 0.1 molar saline solution and
filled into a chromatographic column. The elution of the
column takes place by means of a linear sodium chloride
gradient in the range of 0.1 to 0.8 moles/1 over an entire
elution volume of 2 liters. 100 fractions of a volume of 2 ml
each are collected, and the fractions showing a positive color
reaction with dimethylmethylene blue (DN~IB) of the company
Fluka are united. The solution is narrowed down at 26.7 hPa
(20 torrs) and 40°C and dialysed against water. The dialysate
is set to a volume of 100 ml and a concentration of 0.1
mmoles/1 of calcium acetate and 0.1 moles/1 of sodium acetate,
titrated with acetic acid to pH 7, 1 U of heparinase I,
heparinase II and heparinase III are added, respectively, and
incubation takes place at 37°C for 15 hours.
After dialysing against water and narrowing down under water
jet vacuum, the resulting solution is again applied onto a
column with 10 ml of DEAE Sephadex A50 of the company
Pharmacia Biotech. and eluted as described above. The DN~IB
positive gradient fractions are dialysed, narrowed down under
water jet vacuum to a volume of 1 ml and chromatographed on a
column for preparative gel filtration (60 cm x 2 cm) using a
'14
Sepharose CI-4B gel of the company Pharmacia biotech. 60
fractions of a volume of 2 ml each are collected, detected
with DMMB, and the positive fractions are united. After
repeated dialysis and lyophilisation, the cleaned leucocyte
surface proteo-chondroitin sulfate will be obtained.
3.) Isolation of heparaa sulfate/chondroitin sulfate mixture
from omentum:
One kilogram of fresh bovine omentum is washed with a 0.9 %
NaCI solution, freeze-dried, ground, and degreased with 1 liter
of acetone by stirring over night at room temperature. After
filtering and drying, the resulting powder is suspended in a 6
molar urea solution and stirred over night at room
temperature. After centrifugation at 3000 g for one hour, the
mucous supernatant is decanted, cooled to 4°C, mixed with the
same volume of 1 molar NaOH of a temperature of 4°C, and
incubated for 15 hours at 4°C. Subsequently, neutralization
with dilute HCI, dialyzing against water and centrifugation for
1 hour at 3000 g takes place, and the supernatant is
decanted. In the supernatant, 100 ml of DEAF Sepharose CL-6B
ion exchanger gel of the company Pharmacia Biotech are
suspended and sedimentated. The gel loaded in this way is
still washed three times in a 0.1 molar sodium chloride
solution and filled into a chromatographic column. The elution
of the column takes place by means of a linear sodium chloride
gradient in the range of 0.1 to 0.8 moles/1 over an entire
elution volume of 2 liters. 200 fractions of a volume of 10
ml each are collected, and the fractions showing a positive
color reaction with dimethylmethylene blue (D~) are united.
The solution is narrowed down at 26.7 hPa (20 torrs) and 40°C
and dialyzed against water. Under water jet vacuum, again,
narrowing down takes place to a volume of 5 ml, and
chromatographing is carried out on a column for preparative
gel filtration (60 cm x 5 cm) using a Sephacryl S-300 gel of
CA 02363119 2001-08-24
CA 02363119 2005-09-29
IS
the company Pharmacia Biotech.. 60 fractions of a volume of 10
ml each are collected, detected with DNll4IB, and the positive
fractions are united. After repeated dialysis and
lyophilisation, the purified mesothelial-cell-surface
glycosamino glycan mixture will be obtained.
4.) Isolation of mesothelial cell surface chondroitin sulfate
from tissues rich in mesothelial cells:
One kilogram of fresh bovine kidneys are washed with a 0.9 %
NaCI solution, freeze-dried, ground, and degreased with 1 liter
of acetone by stirring over night at room temperature. After
filtering and drying, the resulting powder is suspended in a 4
molar guanidinium chloride solution and stirred over night at
room temperature. After centrifugation at 3000 g for one hour,
the mucous supernatant is decanted, cooled to 4°C, mixed with
the same volume of 1 molar NaOH of a temperature of 4°C, and
incubated for 15 hours at 4°C. Subsequently, neutralization
with dilute HCI, dialyzing against water and centrifugation for
1 hour at 3000 g takes place, and the supernatant is
decanted. In the supernatant, 100 ml of DEAF Sephacel~ ion
exchanger gel are suspended and sedimentated. The gel loaded
in this way is still washed three times in a 0.1 molar sodium
chloride solution and filled into a chromatographic column.
The elution of the column takes place by means of a linear
sodium chloride gradient in the range of 0.1 to 0.8 moles/1
over an entire elution volume of 2 liters. 200 fractions of a
volume of 10 ml each are collected, and the fractions showing
a positive color reaction with DMMB are united. The solution
is narrowed down at 26.7 hPa (20 torrs) and 40°C and dialyzed
against water. The dialysate is set to a volume of 100 ml and
a concentration of 0.1 mmoles/1 of calcium acetate and 0.1
moles/1 of sodium acetate, titrated with acetic acid to pH 7,
1 U of heparinase I, heparinase II and heparinase III are
a
y6
added, respectively, and incubation takes place at 37°C for 15
hours.
After dialysing against water and narrowing down under water
jet vacuum, the resulting solution is again applied onto a
column with 10 ml of DEAF Sephacel and eluted as described
before. The DN~iB positive gradient fractions are analysed,
narrowed down under water jet vacuum to a volume of 1 ml and
chromatographed on a column for preparative gel filtration (60
cm x 5 cm) using a Sephacryl S-300 gel. 60 fractions of a
volume of 10 ml each are collected, detected with DN~iB, and
the positive fractions.are united. After repeated dialysis and
lyophilisation, the purified mesothelial cell surface
chondroitin sulfate will be obtained.
5.) Immobilization of mesothelial cell surface chondroitin
sulfate with (N-cyclohexyl-N'-2-morpholinoethyl)carbodiimide
methyl tosylate (CME-CDI) onto functional cellulose
surfaces:
100 mg of cellulose membrane are added to a 2 per cent
solution of 3-aminopropyl-triethoxy silane in ethanol/water
(50:50) and stirred for 24 hours at 45°C. Subsequently, the
membranes are washed with a lot of water and are dried. The
membranes treated in this way are immersed into a solution of
1 mg of mesothelial cell surface chondroitin sulfate in 80 ml
of 0.1 molar 2-(N-morpholino)ethane sulfone acid buffer pH
4.75. Over a period of 6 hours at 4°C, 200 mg of (N-
cyclohexyl-N'-2-morpholinoethyl)carbodiimide methyl tosylate
(CME-CDI) of the company Sigma are added in portions of 10 mg
and are further stirred over night at 4°C. Subsequently,
stirring for 2 hours in a 4 molar NaCl solution, washing with
a lot of water and drying in the fresh air takes place.
6.) CNCI immobilization of sphingoglycolipid onto glass:
CA 02363119 2001-08-24
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17
A glass, for example a cover glass for microscopy, is stirred
for 6 hours in 5 ml of chromosulfuric acid. Subsequently,
washing with a lot of water, air-drying and heating to 50°C in
15 ml of dioxane takes place. Subsequently, 2.5 ml of a 2
molar N,N'-diisopropylethylamine solution in dioxane are added
and stirred for 30 minutes. Subsequently, 2.5 ml of a 1 molar
CNCI solution in dioxane are added and stirred for further 2
hours. Subsequently, washing takes place first with dioxane,
then with dioxane/water and finally with pure water. The glass
modified in this way i~s inserted into 20 ml of a solution of 1
mol/1 of ethylenediamine and 0.1 moles/1 of NaHC03,
subsequently heated to 50°C, and stirred for 72 hours at this
temperature. Subsequently, 0.1 mg of sphingoglycolipid of
human erythrocytes are dissolved in 20 ml of 0.1 molar NaHC03
and stirred for 110 hours at 60°C together with the
substituted glass. Subsequently, 2.5 ml of ethanolamine are
added and stirred for further 30 minutes. The coated glass is
washed with a 4 molar NaC) solution and subsequently washed
with a lot of water and dried in the air.
7.) Immobilization of erythrocyte plasma membrane heparan
sulfate onto the oxide layer of nickel, titanium, aluminium
or similar metals:
The metal workpiece is cleaned for four hours in an ultrasonic
bath with hot water, washed with acetone and degreased for one
hour in a Soxhlet extractor with chloroform. The workpiece
cleaned in this way is dried and immersed into a 0.01 - 0.1
molar solution of w-hexadecenyltrichlorosilane in bicyclohexyl
for 2-15 minutes under stirring, washed two times with
chloroform and water, and extracted for 15 minutes with
chloroform in the Soxhlet extractor. The workpiece is immersed
into a solution of 2 ml of acetone and 100 mg of IQ~In04 in 18 ml
of water at 0°C for 45 minutes, and a C02 stream is passed
CA 02363119 2001-08-24
n i r
therethrough. Subsequently, it is immersed for 15 seconds into
a 20% solution of sodium bisulfite in water, washed with water
and dried.
The workpiece is stirred over night in a solution of 29.25 g
of paratoluyl sulfonyl chloride in 900 ml of acetone and 180
ml of pyridine at 40°C. Subsequently, the workpiece is washed
with water and methanol and stirred for 40 hours at 60°C in a
solution of 1 mmol/1 diaminododecane in 1 liter of
dimethylformamide. Subsequently, the workpiece is successively
washed with water, 1 mol/1 soda solution, 1 mmol/1
hydrochloric acid and water. The workgiece prepared in this
way is stirred for 90 minutes in a borate buffer solution
(sodium tetraborate 0.065 moles/1, pH 9.5). Finally, stirring
takes place over night in a solution of 0.3 g of 4-azido-1-
fluoro-2-nitrobenzene in one liter of ethanol at 37°C. 0.5 g
of erythrocyte plasma membrane heparan sulfate are dissolved
in one liter of a 0.1 molar 2-(N-morpholino)ethane sulfone
acid-(MES)-buffer pH 4.75 and stirred with the workpiece at
4°C for 48 hours. The erythrocyte plasma membrane heparan
sulfate is covalently immobilized by illumination for 10
minutes by means of a high-pressure mercury lamp. After
washing with a 4 molar saline solution for 40 minutes, the
workpiece is washed with water and subsequently dried.
8.) Photochemical immobilization of leucocyte plasma membrane
chondroitin sulfate onto cellulose:
3 g of cellulose membrane are allowed to swell in a 4 molar
NaOH for 2 hours, washed three times with water, once with
water/acetone and once with acetone. The cellulose activated
in this way is stirred~over night in a solution of 29.25 g of
paratoluyl sulfonyl chloride in 900 ml of acetone and 180 ml
of pyridine at 40°C. Subsequently, the cellulose membrane is
washed with water and methanol. The resulting esterified
cellulose membrane is now stirred for 40 hours at 60°C in a
CA 02363119 2001-08-24
. . , l9 ,
solution of 1 mmoles/1 of diaminododecane in--1 liter of
dimethylformamide. Subsequently, the membrane is successively
. washed with water, 1 mole/1 of soda solution, 1 mmol/1 of
hydrochloric acid and water. The amino cellulose obtained in
this way is stirred for 90 minutes in a borate buffer solution
(sodium tetraborate 0.065 molar, pH 9.5). Finally, the
membrane is stirred in a solution of 0.3 g of 4-azido-1-
fluoro-2-nitrobenzene in one liter of ethanol over night at
37°C. 0.5 g of leucocyte surface chondroitin sulfate are
dissolved in one liter of a 0.1 molar 2-(N-morpholino)ethane
sulfone acid buffer pH 4.75 and stirred with 2.5 g of the
azido cellulose prepared as described above at 4°C for 48
hours. The leucocyte surface chondroitin sulfate is covalently
immobilized by illumination for 10 minutes by means of a high-
pressure mercury lamp. After washing with a 4 molar saline
solution for 40 minutes and water, the cellulose membrane is
dried.
9.) Immobilization of glycophorin A with glutardialdehyde onto
silicone:
To 1 g of silicone film, 20 ml of water and 2 ml of
3-aminopropyl triethoxy silane are added, and the pH value is
set to 3.5. Subsequently, heating for 2 hours to 75°C, washing
with water and drying takes place. To the resulting amino-
group containing silicone, a 2.5 per cent solution of
glutardialdehyde in a 0.05 molar sodium phosphate buffer is
added, and it is set to pH 7. After stirring for 60 minutes at
room temperature, the activated silicone produced in this way
is reacted with a 0.1% solution of glycophorin A (Sigma) under
stirring for 2-4 hours and is washed with water.
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' ~ r
10.1 Immobilization of erythrocyte plasma me~abrane heparan
sulfate onto polyvinyl chloride (PVC):
0.5 g of iron-II-sulfate, 100 ~.1 of concentrated sulfuric acid
and 2 ml of methacrylic acid are dissolved in 250 ml of water.
125 mg of sodium disulfite and 125 mg of potassium
peroxodisulfate are added to this solution. Subsequently, the
solution is pumped for 2 hours at room temperature through a
ring-shaped PVC tube having a length of 1 m and an inner
diameter of 3 mm. The graft polymerization taking place
thereby is stopped by adding 100 mg of hydroquinone.
Subsequently, the tube is thoroughly washed with water. A
solution cooled to 4°C of 250 mg of CME-CDI (N-cyclohexyl-N~-
2-morpholinoethyl)carbodiimide methyl tosylate in 250 ml of a
0.1 molar 2-(N-morpholino)ethane sulfone acid buffer pH 4.75
is pumped through the tube in a circle at 4°C for 30 minutes.
The tube activated in this way is washed with a 0.1 molar 2-
(N-morpholino)ethane sulfone acid buffer pH 4.75.
Subsequently, a solution of 1 mg of erythrocyte plasma
membrane heparan sulfate in a 0.1 molar 2-(N-morpholino)ethane
sulfone acid buffer pH 4.75 is pumped through the tube in a
circle at 4°C for 15 hours.
Finally, the tube is washed with a 4 molar saline solution and
subsequently with water.
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