U.S. patent application number 14/292238 was filed with the patent office on 2014-09-18 for process for the ultrapurification of alginates.
This patent application is currently assigned to GH Care, Inc. d/b/a Altucell, Inc., GH Care, Inc. d/b/a Altucell, Inc.. The applicant listed for this patent is GH Care, Inc. d/b/a Altucell, Inc., GH Care, Inc. d/b/a Altucell, Inc.. Invention is credited to Giuseppe Pietro Pio Basta, Riccardo Calafiore.
Application Number | 20140275519 14/292238 |
Document ID | / |
Family ID | 40290324 |
Filed Date | 2014-09-18 |
United States Patent
Application |
20140275519 |
Kind Code |
A1 |
Basta; Giuseppe Pietro Pio ;
et al. |
September 18, 2014 |
PROCESS FOR THE ULTRAPURIFICATION OF ALGINATES
Abstract
A method for obtaining solutions of non-structurally modified
salts of alginate, with endotoxin content less than 20 EU/g
includes obtaining an alginate solution having a concentration
ranging from 1.6 to 2.0% by weight by adding commercial grade
alginate powder to a saline solution. The alginate solution
obtained is filtered on at least one hydrophilic filter, and a
filtered alginate solution is recovered. The filtered alginate
solution obtained is further filtered on a charge-modified
hydrophobic filter, and an alginate solution having an endotoxin
content less than 20 EU/g is recovered.
Inventors: |
Basta; Giuseppe Pietro Pio;
(Perugia, IT) ; Calafiore; Riccardo; (Perugia,
IT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GH Care, Inc. d/b/a Altucell, Inc. |
Dix Hills |
NY |
US |
|
|
Assignee: |
GH Care, Inc. d/b/a Altucell,
Inc.
Dix Hills
NY
|
Family ID: |
40290324 |
Appl. No.: |
14/292238 |
Filed: |
May 30, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
12863912 |
Jul 21, 2010 |
8765937 |
|
|
PCT/IB09/50221 |
Jan 21, 2009 |
|
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14292238 |
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Current U.S.
Class: |
536/127 |
Current CPC
Class: |
C08L 5/04 20130101; A61P
3/10 20180101; C08B 37/0084 20130101 |
Class at
Publication: |
536/127 |
International
Class: |
C08B 37/00 20060101
C08B037/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 23, 2008 |
IT |
RM2008A000037 |
Claims
1. A method for obtaining solutions of non-structurally modified
salts of alginate, with endotoxin content less than 20 EU/g,
comprising: a. obtaining an alginate solution having a
concentration ranging from 1.6 to 2.0% by weight by adding
commercial grade alginate powder to a saline solution while
controlling pH; b. filtering the alginate solution obtained from
step a) on at least one hydrophilic filter and recovering a
filtered alginate solution; and c. filtering the filtered alginate
solution obtained from step b) on a charge-modified hydrophobic
filter and recovering an alginate solution having an endotoxin
content less than 20 EU/g.
2. The method according to claim 1, wherein said alginate powder is
sodium alginate powder.
3. The method according to claim 1, wherein said alginate has a
composition comprising 52.26% M and 47.74% G, corresponding to an
M/G ratio of 1.093.
4. The method according to claim 1, wherein said step of filtering
on at least one hydrophilic filter is carried out on three
hydrophil filters of cellulose acetate hydrophils.
5. The method according to claim 5, wherein the first one of said
three filters has a pharmaceutical grade 30 and a nominal pore size
of 2 .mu.m, the second one of said three filters has a
pharmaceutical grade 60 and a nominal pore size of 2 .mu.m and the
third one of said three filters has a pharmaceutical grade 90 and a
nominal pore size of 2 .mu.m.
6. The method according to claim 1, wherein said hydrophobic filter
is a charge-modified Nylon filter.
7. The method according to claim 7, wherein said hydrophobic filter
is a Nylon 66 filter having a positive electric charge.
8. The method according to claim 1, wherein the saline solution of
alginate includes sodium alginate.
9. The method according to claim 8, wherein the sodium alginate is
employed for parenteral use in human transplants.
10. The method according to claim 9, wherein the sodium alginate is
formed into alginate/polyornithine microcapsules to be used in
human cell transplants.
Description
TECHNICAL FIELD
[0001] The present invention relates to a process for the
ultrapurification of alginates, in particular intended for
microencapsulation in human cell transplants. The process is
advantageously applied to the purification of the starting powder
of pharmaceutical grade sodium alginate, removing endotoxins and
endogenous pyrogens, yet keeping unchanged the molecular structure
of the product.
RELATED ART
[0002] Sodium alginate (AG) is a polysaccharide extracted from some
seaweeds, in particular Macrocystis pyrifera, present above all
along the western coasts of the Pacific, which finds wide use in
various fields, biotechnology included, The biopolymer salts, whose
purification is subject-matter of the present invention, are
water-soluble polysaccharides that are either spontaneously exuded
by, or are extracted from living vegetal organisms. In fact,
alginates are salts of alginic acid, which shows a copolymer
structure Dmannuronic acid (-M-), and -L-guluronic acid (-G-)
units), These units form polymer or dimer blocks MM or GG that at
times alternate in the molecular pattern. Alginates molecular
arrangement and composition are determined primarily by the source
from which they are obtained. For example, the most commonly used
alginates derive from brown seaweeds, and in particular products
deriving from Macrocystis pyrifera have an M/G ratio equal to
1.56:1, while those from Laminaria Hyperborea have an M/G ratio
equal to 0.45. Monovalent salts (Na, K) of alginate are typically
water soluble, unlike divalent salts (Ba, Ca) or polyvalent salts
(Fe, Al) that are found in the form of gels or solids.
[0003] AG has been used for years in food and pharmaceutical
industries for the production, respectively, of fruit jellies and
excipients for some classes of drugs (e.g., antiacids, etc.).
However, commercially available sodium alginate is not sufficiently
purified for special applications, such as applications in human
transplants, where strict and internationally recognized quality
control criteria are required, e.g. the guidelines of the Ministry
of Health or those of U.S. Pharmacopeia.
[0004] Alginate is commercially available both as raw extract to be
purified and as partially purified solution. The chemical
composition of alginate powder is described in terms of fractions
(F.sub.G or F.sub.M) and M/G ratio. The product AG KELTONE LVCR,
for example, has an endotoxin level in the range from about 30,000
EU/g to about 60,000 EU/g that, as such, makes it unsuitable for
parenteral applications requiring an endotoxin level of no more
than 100 EU/g, though lower levels are preferable.
[0005] As a consequence of this, and before AG KELTONE LVCR may be
used parenterally, the level of endotoxin must be drastically
reduced.
[0006] For over 20 years, AG has been employed in the preparation
of microcapsules containing hybridoma cells for the production of
monoclonal antibodies (Damon Biotech, Inc.) as well as for
protection of pancreatic islands (insulae) transplants from host
rejection reaction (U.S. Pat. No. 4,683,092). The first
microcapsular transplant protocols in diabetic rodents and higher
mammals clearly highlighted purity of employed material to be
fundamental for success of the transplant itself. The main
contaminant, possibly responsible for microcapsular transplant
failure, is represented by bacterial lipopolysaccharide endotoxins,
pyrogenic materials present in the membrane of gram negative
bacteria. Such substances are resistant to most sterilization
systems (e.g., autoclave). Techniques such as gamma irradiation or
dry heat sterilization are able to destroy endotoxins, but can
damage the materials or products to be sterilized. Moreover, their
generally low molecular weight (10-20 Kd) does not allow removal
thereof by standard filtration processes. In any case, it should be
taken into account the need to obtain a sterile, as well as
endotoxin-free product, in order to avoid the risk of secondary
contamination. Lastly, materials which are to be introduced
parenterally in the human body must have an endotoxin content lower
than 100 EU/g, though it would be preferable to have a level lower
than 50 EU/g. In case of AG use for the preparation of
microcapsules containing pancreatic islands, for transplant
purposes, endotoxin presence may invalidate capsule-provided
immunoprotection, fostering the onset of a serious inflammatory
reaction.
[0007] The need to obtain an ultrapurified product (virtually
"endotoxin-free") has fostered in the last years the development of
some purification methods, which however are unable to meet all
industrial and safety demands. Some purification methods envisaging
the W use of ion exchange resins with or without addition of
polymyxin-b or filtration on cellulose acetate filters and further
membrane dialization had the drawbacks of allowing no relevant
reduction of endotoxin levels (about 70-80 EU/g), being excessive
in cost and not applicable to the production of bulk quantities of
AG owing to a remarkable loss of mass of the starting product,
virtually indispensable to the start of protocols for clinical use,
and, in the case of chloroform use, of exhibiting a difficult
removal of said solvent, potentially toxic even in modest amounts.
For example, in the technique employing AG precipitation in ethanol
and subsequent extraction with chloroform, in order to obtain 1
liter of end product it is necessary to start from about 10 liters
of AG. Moreover, a low yield of the product implies that different
AG batches may have non-homogeneous features, certainly unsuitable
for preparing products to be used in clinical protocols, for which
technologies reproducible on a large scale are essential. U.S. Pat.
No. 6,451,772, starting from raw alginate, substantially provides
filtration (and/or use of ion exchange resins) on polypropylene
filters and subsequent filtrate precipitation with organic
solvents. Main limitations of this process are represented by
[0008] 1. the excessive cost of the materials, with respect to the
end volumetric yield in terms of alginate produced, yield which
however remains unsatisfactory, [0009] 2. the fact that the
described methodology has excessive variability and does not allow
the systematic availability of the product itself, though
occasionally alginate with a sufficiently low endotoxin content is
obtained; and [0010] 3. the modification effect on alginate
structure caused by solvent use.
SUMMARY
[0011] It was now surprisingly found that replacing the
precipitation operation with a filtration on a cartridge with
charge-modified filtration membrane allows to obtain AG [0012] 1.
of very high and constant purity degree (endotoxin content
.ltoreq.20 EU/g) [0013] 2. ultrapurified on a large scale, [0014]
3. obtained without use of solvent, and therefore [0015] a. without
modification of the chemical structure of alginate [0016] b. and
without alginate contamination, so as to be accepted by protocols
for parenteral use.
[0017] It is therefore subject-matter of the present invention a
process for the obtainment of solutions of salts of alginate not
structurally modified, with endotoxin content not higher than 20
EU/g, comprising the following steps: [0018] a. addition of
commercial grade alginate powder to a saline solution, until
obtaining an alginate solution having a concentration ranging from
1.6 to 2.0% by weight and pH adjustment in the range 7.4-7.6 [0019]
b. filtration of the solution obtained from step a) on at least one
hydrophilic filter and recovery of the solution obtained
[0020] characterized in that the solution obtained from step b) is
subjected to filtration on a hydrophobic filter and the obtained
solution is recovered.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0021] According to the invention, an alginate used is sodium
alginate having a composition comprising 52.26% M and 47.74% G,
corresponding to an M/G ratio of 1.093. Advantageously, as alginate
the product Keltone.RTM. LVCR was used.
[0022] The filtration of step b) preferably occurs by using three
filters of cellulose acetate hydrophils, of which advantageously
the first one has a pharmaceutical grade 30 and a nominal pore size
of 2 .mu.m, the second one a pharmaceutical grade 60 and a nominal
pore size of 2 .mu.m, and the third one a pharmaceutical grade 90
and a nominal pore size of 2 .mu.m.
[0023] The second filtration makes use of a hydrophobic filter; it
is advantageously preferred a charge-modified nylon filter, in
particular a nylon 66 filter having a positive electric charge.
[0024] According to the invention, from the pharmaceutical grade AG
powder, after molar dilution and multiple filtration, an end
product is obtained (both in the form of solution and powder) whose
endotoxin content does not exceed 20 EU,/g, in full compliance with
the above-stated quality control criteria. The end product of the
present inventors, usually available in 1.8% (w/v) solution,
appropriately stored in a light-protected environment and at a
temperature of 4.degree.-6.degree. C., is stable over time for
about 5 years with a virtually absent protein content
(<0.4%--another U.S. FDA bioinvisibility standard).
[0025] The invention exploits the chemical structure of
lipopolysaccharide endotoxins (LUPS) consisting of two portions,
one hydrophilic (polysaccharide) and one hydrophobic (lipid).
According to the invention, at the end of the ultrafiltration
process (positively charged) nylon filters were employed, able to
selectively bind the (hydrophobic) lipid portion of endotoxins,
retaining them without altering and/or damaging AG structure.
Adding the fourth filter, besides obviating further manipulations,
required according to the prior art, guarantees a reproducible and
simple result. Moreover, the chemical structure of the product is
not altered, an aspect that had been highlighted in the
above-mentioned U.S. patent. This is incontrovertibly demonstrated
by NMR spectra of the product showing, both in the case of
Deuterium and of Carbon, that there are no structural nor molecular
variations, not even minimal ones, between the (unpurified)
starting product and the (ultrapurified, clinical grade) end
product.
[0026] The process according to the invention allows obtaining
about 50% of the starting product, in comparison with the 10% that
may be calculated as recovered amount with the other
methodologies.
[0027] The process of the present invention is simple to carry out,
with limited manual interventions, therefore less at risk of
contamination than the previous known ones, as it entails only the
assembly of the suitable filter in the sanitary container
("housing") and the connection to the pumping system, down to the
collecting one. The entire methodology may easily be conducted
under a class II laminar-flow biological hood. Protein content of
these samples ranges from 0 to 0.016 mg/ml. These data were
calculated on values obtained in 21 different filtration processes
accomplished in 2 years, as shown in the following Table 1.
Table 1
[0028] Protein content in various samples obtained according to the
process of the invention Average: 0.090846
TABLE-US-00001 Purification Proteins (internal number) (mq/ml) 1
0.0072 2 0.0087 3 0.0179 4 0.0095 5 0.0116 6 0.0121 7 0.0077 8
0.0062 9 0.0098 10 0.0118 11 0.0125 12 0.0124 13 0.0138 14 0.0089
15 0.0120 16 0.0131 17 0.0088 18 0.0063 19 0.0110 20 0.0157 21
0.0079
[0029] The process of the invention comprises two steps: [0030] 1.
Dissolving the AG powder in sodium chloride, controlling the pH,
and passing through three hydrophilic filters. [0031] Preferably,
as hydrophilic filtering material it is employed cellulose acetate,
or other commercially available filters, for example "zetaplus"
grade Cuno filters having a pore size ranging from 1 microns to 0.1
microns, with a wide filtering surface such as that employed by the
present inventors, which offers the advantage of reducing
filtration pressure without altering the product and of removing
cell fragments and the so-called "inert" microparticles. The
product thus obtained is sterile, with an endotoxin content anyhow
higher than 100 EU/g; [0032] 2. the second step, supporting its
originality, is the removing of residual endotoxins by a
hydrophobic nylon filter having a positive electric charge, in
order to bind the negative portion of the lipopolysaccharide
preventing precipitation on solvent or of other techniques,
obtaining a product ready for various uses, having an endotoxin
content always lower than 30 EU/g.
[0033] The present invention finds application in the field of
transplantation biotechnologies, in particular in the production of
AG microcapsules suitably coated with polyamino acids and diluted
AG, which were demonstrated to immunoprotect transplants of islands
from host immune system cells. For several years now the inventors
have been studying the transplant of microencapsulated pancreatic
islands for therapy of type 1 diabetes mellitus (insulin-dependent,
or T1DM) and this research activity is amply documented by
scientific publications, mostly international ones. Moreover,
thanks to the purity and high biocompatibility of the materials
employed, the present inventors have been authorized by the Italian
Istituto Superiore di Sanita to operatively begin a phase I
clinical study related to transplant of microencapsulated human
pancreatic islands in patients with T1DM, not pharmacologically
immunosuppressed. From results obtained hereto it emerges that the
microcapsules obtainable according to the invention are highly
biocompatible, and the effectiveness of their product has been
assessed by various international research laboratories.
EXAMPLES
[0034] For the preparation of the solution of sodium alginate,
ultrapure and having an endotoxin level no higher than 20 EU/g, the
following materials are needed: [0035] Pyrex beaker, [0036] Pyrex
graduated cylinders, [0037] Magnetic stirrer, [0038] Silicone tube
HW 155 inner diameter 5, outer diameter 8, [0039] Peristaltic pump,
[0040] Sterile pipettes, [0041] Certified endotoxin-free sterile
bottles, [0042] 30, 60 and 90 pharmaceutical grade cellulose
acetate filters with filtration coadjuvant comprised of diatomite
and perlite, 20'' height and 12'' diameter [0043] Filters with
charge-modified Nylon 66 filtration membrane, pore size 0.2 .mu.m,
20'' height and 8'' diameter. [0044] AISI316 stainless steel
sanitary container for 20'' filtration cartridge, 12'' diameter.
[0045] AISI316 stainless steel sanitary container for 20''
filtration cartridge, 8'' diameter.
[0046] The methodology of preparation of the solution of sodium
alginate, ultrapure and with an endotoxin level no higher than 20
EU/g provides the following steps:
Sterilization of "Housings", Filters and Material of Use
[0047] Glass containers (graduated beaker, graduated cylinder,
bottles, etc.), connecting silicone tubes, AISI316 stainless steel
sanitary containers, referred to as "Housings", and any other
material (magnetic anchors, glass rods, etc.) used during the
procedure for the preparation of the alginate solution and during
the filtration steps are treated for 24 h with an 1% etoxate
solution (E-Taxa-Clean.RTM., Catalog Number E9029. SigmaAldrich,
Milan, Italy), then accurately washed with deionized water and
lastly autoclaved at 120.degree. C. for 1 hr. The 30, 60 and 90
pharmaceutical grade cellulose filters, all with 0.2 .mu.m in pore
size, and the cartridge with charge-modified Nylon 66 filtration
membrane, 0.2 .mu.m pore size, are autoclaved, separately from the
Housing, at 120.degree. C. for 1 h.
Preparation of 1.8% AG Solution
[0048] AG (the sodium salt of alginic acid (E400)) used is
Ketone.RTM. LVCR (Kelco) having a low degree of viscosity and
provided in form of ultrapure powder by the producer:
Monsanto-Kelco (20 N Wacker Dr, Chicago Ill. USA). The chemical
composition of alginate powder is described in terms of fractions
(FG or FM) and MIG ratio, and the alginate used by the present
inventors has an M percent of 52.26% and a G percent of 47.74% and
the M/G ratio is 1.093, determined through NMR (Nuclear Magnetic
Resonance) analysis. All the procedure for the preparation of the
1.8% alginate solution is carried out under a class II laminar-flow
biological hood. After weighing, the alginate powder is placed in a
beaker and physiological solution (0.9% NaCl) is added slowly, to
avoid clotting (the physiological solution used is sterile,
apyrogenic and specific for injectable preparations) under bland
stirring attained by use of a magnetic stirrer and a magnetic
anchor, until obtaining a homogeneous solution.
Filtration Systems (Housing) Assembly
[0049] At the moment of use, the suitable filter is inserted in the
housing, all the procedure is carried out under biological hood,
and said filter is closed and assembled on the specific support.
Silicone tubes are fastened at the outlet and inlet of the housing.
The tube at the inlet of the filtration apparatus is hooked to a
peristaltic pump, whereas the free end is immersed in the beaker
containing the alginate solution. The free end of the silicone tube
at the outlet of the filtration system is placed in a sterile
collection bottle.
Filtration
[0050] The solution is subjected to 4 different filtration steps,
which are all performed by using the "Housing" filtration system
and without breaks.
[0051] The first step provides for the solution to be filtered
through a capsule of 30 pharmaceutical grade cellulose fiber
(nominal pore size 2.4 .mu.m). By means of the peristaltic pump,
the housing is filled with about 7 liters of product, and filtrate
collection is started. Pump rate is set at very low values, such as
to allow a greater interaction between material and filter, and in
fact the pressure inside the housing is held in the neighborhood of
values of 1.5 bar. The first fraction of the filtrate, roughly
quantifiable in 2 liters, is discarded as rich in extractables. The
remainder is collected in plastic bottles, sterile and certified
for endotoxin absence.
[0052] The second filtration step provides for the use of a 60
pharmaceutical grade cellulose fiber capsule (nominal pore size 2
.mu.m). The rate at which the pump is set is, in this case as well,
such as to generate in the filtration system a pressure of 1.5 bar.
Collection occurs in the same type of plastic bottles described
above.
[0053] The third step on cellulose fiber capsule provides for the
use of a 90 pharmaceutical grade capsule (nominal pore size 2
.mu.m). The rate at which the pump is set is, in this case as well,
such as to generate in the filtration system a pressure of 1.5 bar.
Collection occurs in the same type of plastic bottles described
above.
[0054] The fourth and last filtration step provides for a cartridge
with a charge-modified Nylon 66 filtration membrane, pore size 0.2
.mu.m. In this last case the pressure generated by the peristaltic
pump is held in the neighborhood of 0.3-0.5 bar, by acting on the
control of the rate of the pump itself. The filtrate is now
collected in glass bottles pretreated for endotoxins and sterilized
in autoclave at 120.degree. C. for 1 h.
End Product Evaluation
Example 2
[0055] Aliquots of the obtained product are tested for: endotoxin
presence, by taking a suitable aliquot and sending it to a company
specialized in endotoxin dosage by Limulus method (Lonza Verviers,
SPRL), for protein content by Bradford method, for pH value at
+4.degree. C. and at +20.degree. C., using the micrometric method,
and to confirm its chemical composition and the related monomer
fractions by NMR analysis. Endotoxin content is found lower than 20
EU/g. Heavy metal presence and product sterility are assessed
through standard protocols.
[0056] The present invention finds application in the field of
transplantation biotechnologies. In particular, in the case of the
present inventors, AG microcapsules suitably coated with polyamino
acids and diluted AG were demonstrated to immunoprotect cells from
the immune system of the receiver. For several years now the
inventors have been studying in their laboratory the transplant of
microencapsulated pancreatic islands for therapy of type 1
(insulin-dependent) diabetes mellitus and this research activity is
amply documented by scientific publications, mainly international
ones. Moreover, thanks to the purity and high biocompatibility of
the materials employed, the present inventors have been authorized
by the Istituto Superiore di Sanita to operatively begin a phase I
clinical study related to transplant of microencapsulated human
pancreatic islands in diabetic receivers, not pharmacologically
immunosuppressed. From results obtained hereto, it emerges that the
microcapsules of the present inventors are highly biocompatible,
and the effectiveness of their product has been assessed in various
international research laboratories.
[0057] Capsules and artificial extracellular matrices for growth
and differentiation of various cellular strains STANDARD OPERATIVE
PROCEDURE (SOP) for the preparation of alginate/polyornithine
(AG/PLO) microcapsules using sodium alginate produced according to
the present invention
Reagents
[0058] sterile and apyrogenic physiological solution; [0059] 1.2%
CaCl.sub.2 solution in distilled water; [0060] solution of 55 mM
Na-citrate in distilled water; [0061] 0.12% and 0.06% polyornithine
solutions in physiological solution (the above-mentioned solutions
are sterilized by filtration) [0062] 1.6% NAG, obtained by the
aforedisclosed filtration process; [0063] 0.05% NAG, by 1:10
dilution of the preceding one in physiological solution.
Method
[0064] Islands are washed with physiological solution to remove any
protein present.
[0065] Then, for each ml of pellet 0.5 ml of physiological solution
and 10 ml of 1.6% NAG are added, bringing the suspension to
homogeneity.
[0066] The peristaltic pump is adjusted at 15 ml/min and the air
flow at 5 l/min, initially letting physiological solution flow
through the system.
[0067] In a 250 ml beaker, containing 200 ml of 12% CaCl.sub.2,
there will be collected the alginate microdrops which, by gelling,
will form the island-containing alginate microcapsules. The
distance of the needle from the meniscus of the CaCl.sub.2
solution, equal to about 3 cm, assumes critical importance.
Capsules are left S min in CaCl.sub.2, then 100 ml of 1.2%
CaCl.sub.2 are replaced with physiological solution and left again
for S min. Then, after repeated washings with physiological
solution, the capsules are transferred into a 50 ml falcon. Then,
the following reactants will be added in sequence, in an amount
equal to twice the volume taken up by the capsules, removing each
time what was previously added, stirring and carrying out between a
reactant and the other suitable washings with physiological
solution: [0068] 0.12% polyornithine for 10 min; [0069] 0.06%
polyornithine for 5 min; [0070] 0.1% NAG for 6 min; [0071] 55 mM
Na-citrate for 2 min.
[0072] The repeated washings carry out a two-fold function, as they
allow on the one hand the removal of reactants or cellular debris,
and on the other hand the removal of most of the smaller capsules,
empty or broken, which by being lighter in weight settle more
slowly. Upon ending these treatments, the capsules will be
resuspended in CMRL 1066 medium. Prior to implant the capsules are
washed with physiological solution, in which they will be
resuspended also at the moment of implant.
* * * * *