Microparticles, preparation thereof and applications thereof in biology, particularly in the culture of human diploid cells

These microparticles are formed, at least on their surface, from a reticulated protein. (FIG. 1).

BACKGROUND OF THE INVENTION 
The invention relates to novel microparticles, the preparation thereof and 
the applications thereof in biology, especially in the culture of animal, 
and in particular human, diploid cells, normal or infected for example by 
a virus or a parasite. 
It is known that normal diploid cells which are cells of embryonic origin 
are used for elaborating different products of cellular origin, such as 
interferons, or else for culture of viruses suitable for the manufacture 
of viral vaccines for human use. 
But the production of these cellular elements on an industrial scale is 
difficult due to the requirements for the culture of normal diploid cells. 
In fact, contrary to transformed cells, which are capable of developing in 
suspension in a liquid medium, so with a satisfactory yield, normal 
diploid cells can only multiply on a solid support. 
Single layer culture on a solid support of a large quantity of cells in 
Roux boxes or in rollers requires a large number of containers, which 
constitutes a limiting factor and has proved unusable for commercial 
purpose. Research has been carried out with respect to solid carriers for 
carrying out cultures adaptable to mass culture conditions in fermentation 
tanks. 
Microparticles or beads, in general in the form of microbeads of a diameter 
of about 40 to 200 .mu.m have more particularly attracted attention for 
this purpose. They allow in fact substantially homogeneous cultures to be 
carried out adaptable in principle to culture conditions in a fermenter. 
They have furthermore the advantage of offering a large culture surface, 
which allows an increase in the produced cell yield. 
However, microbeads known up to present do not entirely satisfactorily 
resolve the particular problems of adherence, multiplication, toxicity, 
met with in the culture of normal or infected human diploid cells. 
Now, the work carried out in this field by inventors has established that 
particular microcarriers, especically for the culture of diploid cells 
allowing an extremely satisfactory adherence and growth of cells on their 
surfaces, could be elaborated from some types of proteins used for the 
formation at least of their surfaces. 
The invention relates then to novel microparticles favoring, because of 
their composition and the properties which result therefrom, the adherence 
and the growth of human diploid cells. 
It also relates to a process for preparing these microparticles. 
In accordance with another aspect, the invention relates to the application 
of these microparticles to the culture of cells, more particularly normal 
human diploid cells, and to the culture of infected diploid human cells or 
with the in situ infection thereof in view. 
SUMMARY OF THE INVENTION 
The microparticles, in accordance with the invention, to be designated 
without distinction also by the term microcarriers or else microbeads, are 
characterized in that they are formed from particles of which at least the 
surface is formed by a reticulated protein, this protein being chosen from 
those capable of forming a gel, i.e. a visco-elastic mass giving rise, 
after reticulation, to a fibrous-type mass reticulated into a mesh. 
These particles have advantageously a charge capacity of about 0.5 to 1.8 
meq per gram of dry particles. Preferably, the charge capacity of these 
microbeads is from 0.9 to 1.6 meq/gram and advantageously of 1.+-.0.3 meq 
per gram of dry particles. 
In a remarkable way, the microbeads complying with the above-defined 
characteristics prove particularly efficient as microcarriers for the 
culture of human diploid cells and this, even with aged cellular cultures 
already having undergone several passages, i.e. having given rise to 
several generations. 
In the rest of the description, reference will be more particularly made to 
human diploid cells, considering the quite particular interest which the 
invention assumes for their culture. But it is clear that the invention 
also applies with advantage to cultures of animal diploid cells. 
The protein, present at least at the surface of these microbeads, forms a 
precious carrier for the adherence of cells and the multiplication 
thereof. 
In accordance with a preferred embodiment of the invention, the protein 
entering into the composition of the microbeads and forming at least the 
surface thereof is formed by gelatin. 
It is known that gelatin is a product from the partial hydrolysis of 
collagens. Depending on the method of manufacture thereof, a distinction 
is made between the gelatin obtained by the acid process, having an 
isoelectric point pHi between 7 and 9 and that obtained by the alkaline 
process having a pHi between 4.7 and 5. 
These two types of gelatin may be used within the scope of the invention, 
the gelatin obtained by the alkaline process having the advantage, because 
of its surface tension properties, of spreading out more easily while 
giving rise to a substantially homogeneous surface, more suitable for the 
contemplated biological applications, as microcarriers. 
In another embodiment of the invention, the protein entering into the 
composition of the microbeads is formed by fibronectine. This is a protein 
of the fibrinogen type, having a molecular weight of about 440,000 present 
in plasma and at the surface of different cells. This protein is described 
particularly in Proc. Natl. Acad. Sci. USA, Vol. 76 No. 6 p. 2644-2648, 
June 1979, by S/A/ Santoro and L. W. Cunningham. 
In accordance with the invention, the proteins entering into the 
composition of the microbeads are reticulated. The reticulation agent is 
advantageously chosen from conventional reticulation agents. Use is more 
specially made of a multifunctional agent having at least two aldehyde, 
azo, sulfonic acid, fluoro groups activated by reactive nitro, imine azide 
or chloro groups connected with cyclic structures having a suitable 
resonance. 
Because of its efficiency and its availability, glutaraldehyde is 
advantageously used. 
With the purpose of using the microparticles of the invention as 
microcarriers for cellular cultures, so as to obviate the possible risks 
of toxicity, with respect to the cells, the functional groups of the 
recticulation agent, not engaged in the reticulation action, are blocked. 
The blocking of the functional groups in question is effected 
advantageously with the help of the protein entering into the composition 
of the microbeads. 
In accordance with a variation of the invention, the microbeads are formed 
essentially by the reticulated protein, preferably by the recticulated 
gelatin such as mentioned above, or else by reticulated fibronectine. 
According to another variation of the invention, the microbeads contain the 
reticulated protein at their surface, in the form of a coating. 
Such microballs are preferably formed of particles comprising a coating of 
reticulated protein fixed on an anchoring nucleus. 
An appropriate nucleus is formed from a material having chemical groups 
capable of reacting with the functional groups of the protein of the 
coating while giving rise to a link and having a density allowing the 
particles to be maintained in suspension in a culture medium. 
As preferred material of this type, use is made of reticulated dextran such 
as the one commercialized under the trademark Sephadex by Pharmacia Fine 
Chemicals, Inc. substituted by tertiary or quaternary amino groups, in a 
proportiion such that the charge capacity of the particle before coating 
is of the order of 0.5 to 1.8 meq per gram of dry Sephadex, preferably 
from 0.9 to 1.6 and advantageously 1.+-.0.3 meq per gram of dry Sephadex. 
It will be noted that the charge capacity of the protein-coated particles 
is substantially of the same order of size. 
The amino groups are more specially formed by diethylaminoethyl (DEAE) or 
diethyl-2-hydroxy-propyl-aminoethyl (QAE). 
For preparing the microcarriers of the invention, a solution, 
advantageously aqueous, is formed of a protein having the above-defined 
characteristics, in a concentration for obtaining particles having the 
desired properties considering their biological applications. 
This solution is then treated so as to form particles constituted entirely 
by the protein in question or, according to a variation, comprising this 
protein in the form of a coating. 
To elaborate the protein particles, the protein solution is treated so as 
to form particles, preferably droplets having a diameter of the order of 
40 to 200 .mu.m, particularly by pulverizing it in a bath consisting, for 
example, of a mixture of vegetable oil, n-butyl alcohol and glutaraldehyde 
in suitable concentrations for the mass setting of the droplets. The 
protein solution may also be caused to flow through a capillary tube. 
As for the protein coating, it is advantageously obtained by contacting 
said protein solution with a suspension, preferably aqueous, of particles 
having a diameter of the order of 40 to 200 .mu.m, formed from a material 
capable of forming an anchoring nucleus for the protein coating such as 
defined above. Such a material having a low charge capacity is 
advantageously prepared in accordance with the technique described in U.S. 
Pat. No. 1,777,970. The operating conditions, particularly temperature, 
duration, pH, being chosen depending on the products used, the coating and 
the charge capacity desired. 
Experience shows that a satisfactory coating is obtained by operating at a 
pH close to 7 and at a temperature greater than the ambient temperature, 
advantageously less than 40.degree. C., a suitable reaction time then 
being of the order of 24 hours. To promote contact of the particles with 
the protein, the reaction mixture is advantageously subjected to 
agitation. The protein particles, or particles covered with protein, 
obtained are washed then placed in contact with a solution, also 
advantageously aqueous, of the reticulation agent. 
The optimum temperature, duration and concentration conditions are 
finalized in routine operations so as to obtain the particles having the 
desired characteristics depending on the protein and the reticulation 
agent used. 
As already pointed out, the free functions of the reticulation agent are 
blocked. 
After the reticulation step, the particles are advantageously washed and 
then placed in contact with a solution, preferably aqueous, of the protein 
of the microball, and this under conditions allowing the desired blocking 
to be achieved. 
An electronic microscopic examination of the microbeads thus obtained shows 
that they have a surface having the appearance of a lattice-work. 
Work carried out on these microbeads has shown that, unexpectedly, they 
allow extremely satisfactory adherence and multiplication of normal human 
diploid cells. 
It is also possible to infect, with a virus or a parasite, the cells which 
have grown on the microbeads, which allows viruses, or parasites, to be 
prepared. 
The microbeads of the invention are then advantageously used as 
microcarriers in cellular cultures, more specially of normal or infected 
human diploid cells. 
The invention relates then also to a culture process for human diploid 
cells comprising use of the above-defined microbeads. 
In a preferred embodiment of this process, a sterile suspension of the 
microbeads in question is formed in a medium suitable for the culture and 
the growth of normal human diploid cells. This medium is seeded under 
sterile conditions with the diploid cells, then it is subjected to 
incubation under temperature and duration conditions allowing satisfactory 
multiplication of the cells. 
The seeding step is advantageously carried out by using the human diploid 
cells at the rate of about 10.sup.5 cells per ml of culture medium for 
about 1 to 2 mg of microbeads per ml of culture medium and preferably for 
1.5 to 1.7 and more especially about 1.6 mg of microbeads. 
The seeding step is also advantageously carried out by using the human 
diploid cells at the rate of 2.5.times.10.sup.5 cells per ml of culture 
medium for 4 mg of microbeads per ml of culture medium. 
The culture medium in which the microbeads are in suspension is preferably 
brought up to the temperature which will then be used during the 
incubation step. 
During this incubation step, it is important to offer the cells the maximum 
of surface of the beads. The seeded medium is then advantageously agitated 
to make it homogeneous. It is then left to sediment so as to allow contact 
of the cellular mass with the microbeads with a view to the adhesion of 
cells. Finally, it is slightly agitated to ventilate the cellular 
layer-microbead interface. 
A preliminary treatment of the microbeads used as microsupports in the 
culture process of the invention allows favorably a better cellular 
growth. 
A suitable treatment comprises at least a step for sterilizing the 
microbeads in a phosphate buffer called "PBS". These sterile microbeads 
are then preincubated in complete culture medium to which there is 
possibly added lactalbumin hydrolysate. This culture medium comprises all 
the elements required for the growth of the cells and will be used in the 
incubation step carried out after seeding. 
This preincubation step is advantageously effected at a temperature between 
ambient temperature and about 40.degree. C., preferably at 37.degree. C. 
and, under these conditions, for about 24 hours in the presence of 
lactalbumin hydrolysate. 
It is also preferable, before preincubation, to subject the microparticles 
to a sterilization step. The sterilization may be effected by using as 
suspension medium the washing buffer, to which there is possibly added the 
protein entering into the composition of the microparticles. 
Afterwards, the microparticles are rinsed with PBS and they are 
preincubated in a Dulbecco culture medium containing possibly lactalbumin 
hydrolysate, in the case where the microparticles are subsequently seeded 
with human diploid cells. All these operations take place naturally in a 
sterile way. 
The growth steps effected bring out the precious qualities of the 
microcarriers used in accordance with the invention. 
As is shown by the results given in the following examples, their 
efficiency appears remarkable not only insofar as the rate of adhesion of 
the cells is concerned but also as far as cellular division is concerned. 
It can in fact be seen with interest that the percentage of adhesion of the 
cells on the microcarriers with respect to the initial number of cells 
added to the culture flask is extremely satisfactory and reaches values of 
the order of 80%, 24 hours after seeding. 
This strong adhesion of the cells at the beginning of the culture is 
advantageously followed by high multiplication of these cells on the 
microcarriers. 
The number of cell divisions observed after 5 to 7 days of culture proved 
in fact to be greater than 2. 
Advantageously, the work carried out shows moreover that the cells grown on 
the microcarriers of the invention may be used again in a fermenter having 
a large volume, which shows the viability of the cells. 
Such results make possible the use of the process of the invention 
particularly in the large-scale production of viruses with a view to 
preparing viral vaccines or other cellular products such as interferons.

DESCRIPTION OF THE PREFERRED EMBODIMENTS 
Example 1 
Preparation of Sephadex-DEAE microbeads covered with reticulated gelatin 
These microbeads are prepared using the three following steps (a) to (c), 
in which: 
(a) DEAE groups are grafted onto the Sephadex microbeads, 
(b) the Sephadex-DEAE beads obtained are covered with gelatin, 
(c) reticulation of the gelatin and blocking of the functional groups from 
the reticulation agent are carried out. 
Each of these steps are carried out as follows: 
(a) Grafting of the DEAE groups on the Sephadex microbeads. 
Reticulated dextran microbeads commercialized by the Company PHARMACIA 
under the trademark Sephadex, of the type medium G 50, are allowed to 
swell in water, then they are washed several times with distilled water. A 
suspension of 50 g of these microbeads in 500 ml of distilled water is 
then formed. A 500 ml aqueous solution is prepared containing 0.5 mole of 
diethylaminoethyl chloride and 0.75 mole of NaOH, another DEAE halide, 
such as bromide, and/or another alkaline hydroxide, such as KOH, may be 
used. The pH of this solution is greater than 11. This solution is added 
to the microbead suspension in a reactor thermostatically controlled to 
60.degree..+-.1.degree. C. The mixture is agitated within the medium with 
a stainless-steel propeller having 4 blades placed perpendicularly. The 
reaction is allowed to continue for 30 to 80 minutes depending on the 
tests carried out. The reaction mixture is cooled by adding a volume of 
cold distilled water so as to stop the grafting. 
It is allowed to decant and the matter floating on the surface is removed. 
This operation is repeated several times so as to eliminate the reagents, 
particularly the DEAE, and to obtain a pH equal to 7. 
The charge capacity of the microballs thus obtained is checked. For this 
purpose the chloride anions exchanged with the microbeads are measured. 
The microbeads are washed first of all with a solution of 0.1 N 
hydrochloric acid so as to saturate the exchange sites with Cl.sup.- ions, 
then they are washed with a 10.sup.-4 N solution of HCl, to remove the 
nonfixed choride ions and finally with a 10% w/v solution of sodium 
sulfate so as to displace the chloride anions with sulfate anions. After 
this washing the solution is collected which is titrated with a 1 M silver 
nitrate solution using potassium chromate as indicator. 
The measurements carried out show that the microbeads obtained have a 
charge capacity per gram of dry G 50 Sephadex of about 0.9 to 1.6 meq. 
(b) Coating of the microbeads with gelatin 
To 100 ml of gelatin obtained by the alkaline process, such as the limed 
bone gelatin 240 BLS commercialized by the firm ROUSSELOT, Paris, France, 
at 10% w/v, there is added 200 ml of a suspension containing 10 g of 
microbeads such as obtained in step (a). 
This solution, having a pH close to 7, is maintained at 37.degree. C. for 
24 hours with weak agitation. After 24 hours, the beads are washed several 
times with distilled water to eliminate the excess gelatin. 
(c) Reticulation of the gelatin and blockage of the functional groups from 
the reticulation agent. 
The beads are placed again in suspension in 200 ml of distilled water then 
20 ml of glutaraldehyde in a 25% aqueous solution are added. 
The mixture is kept at ambient temperature for 24 hours and with weak 
agitation. 
After 24 hours, the beads are washed several times with distilled water to 
eliminate the excess glutaraldehyde. The absence of reagent in the 
floating matter resulting from washing is checked by measuring the optical 
density at 280 nm (adsorption wavelength of glutaraldehyde). 
The free functions of the glutaraldehyde are then blocked with gelatin. 
The microbeads are then taken again in 200 ml of distilled water and 100 ml 
of gelatin at 10% are added. 
This solution is maintained at 37.degree. C. for about 10 hours. To 
eliminate the free excess gelatin present in the floating matter, the 
microbeads are then washed (several times) with distilled water. 
In FIG. 1, there is shown an electronic microscope photograph of microbeads 
thus prepared. It can be seen that the recticulated gelatin coating forms 
a fine net at the surface of the microbeads. 
Example 2 
Treatment of the microbeads obtained in Example 1 with a view to their 
application as microcarriers for cellular cultures 
The microbeads are washed first of all 5 to 6 times with PBS. Then a 
suspension of these microbeads in PBS, containing possible 3 to 4% 
gelatin, is placed in an autoclave at 120.degree. C. for 20 minutes, in a 
humid vapor. 
Example 3 
Use of the microbeads treated in accordance with Example 2 as microcarriers 
for cellular culture 
Before proceeding with the seeding step, the microbeads treated in 
accordance with Example 2 are washed with a culture medium called Dulbecco 
medium, prepared in the laboratory, to which lactalbumin hydrolysate is 
added and if necessary antibiotics. This Dulbecco medium is described 
particularly in "International Association of Microbiological Societies 
Permanent Section of Microbiological Standardization", Minutes of the 
Seventh Meeting of the Committee on Cell Cultures, a conference which was 
held in Geneva on Sept. 14, 1970, edited by Professor Hayflick and Dr. 
Perkins, p. 123-124, published in 1971. After the third washing, the 
microbead suspension is incubated at 37.degree. C. for 12 hours. 
At the time of testing, after decantation, a suspension fraction containing 
1.6 to 5 mg/ml is taken and placed sterilely in a 250 to 1000 ml flask of 
the spinner type. The microbeads are left to decant then they are rinsed 
twice with 10 ml of Dulbecco medium to which is added 10% of aseptic calf 
serum coming from Medical and Veterinary Supplies, Slough, GB. The volume 
is completed to 100 ml or 150 ml or 500 ml or 1 liter, depending on the 
volume of the flask. 
The suspension preheated to 37.degree. C. is sterilely seeded with human 
foetal lung cells MRC 5 obtained from the 27th to 32nd passage. 
The cells come from a deep-frozen stock prepared in the laboratory, 
complying with international standards. The cell culture is treated with a 
0.25% trypsin dispersant solution. The cells are counted and added to the 
culture flask in a proportion of about 10.sup.5 cells for 1.6 mg of 
microbeads per ml of suspension. After seeding, the culture medium is left 
at 37.degree. C. for two hours under agitation, then the agitation system 
is started up. 
So as to check the adhesion of the cells, the first day and the growth the 
following days, 1 ml of microball suspension is taken each day. 
The cells are counted as follows. The suspension taken is decanted then 
rinsed once with a trypsin: versene volume for volume mixture 
(1.25.permill.:0.1.permill. final concentrations). Then they are subjected 
to incubation at 37.degree. C. in the presence of 1 ml of the trypsin: 
versene mixture. 
After 5 to 10 minutes, the suspension is subjected to agitation then 
isotonic liquid is added of the same density as the cells. After 
sedimentation of the beads, the cells are counted in the floating matter 
with a Coulter counter sold by Coultronics. The pH of the medium is 
measured and adjusted regularly with a 5.5% bicarbonate solution. The 
cellular growth is estimated in accordance with two parameters, namely: 
(1) percentage adhesion of the cells on the microcarriers with respect to 
the initial number of cells added to the culture flask; 
(2) the maximum number of cells obtained after 5 to 7 days culture, which 
allows the number of cellular divisions to be calculated. 
There is obtained, 18 hours after the subculture cells having adhesion 
percentages on average greater than 30-40% and reaching 80 to 90%. As to 
the number of divisions obtained, it is generally greater than 2 and may 
reach, operating under the conditions described above, values close to 3. 
The trend of the growth curves of the cells on the microbeads of the 
invention shows the appearance of a plateau, which represents the maximum 
density of the cells, about the sixth day after seeding or depending on 
the tests the seventh or eighth day. 
There is shown in FIG. 2 the growth curve of MRC-5 cells on Sephadex G 50 
DEAE microbeads coated with gelatin, having a charge capacity of 1.1 meq 
per gram of dry and nontreated Sephadex G 50. The growth test was carried 
out in a volume of 100 ml with 4 mg/ml of microbeads having undergone the 
pretreatment described in Example 2, without use of gelatin and 
2.5.times.10.sup.5 cells. 
A change of medium was carried out 4 days after the beginning of the test 
so as to eliminate the metabolites always rejected into the medium and 
thus to maintain growth. The growth curve on which there is shown as 
abscissa the duration of the test, expressed in days, and as ordinates the 
number of cell divisions, has the appearance of a normal curve obtained in 
a monolayer culture system. The appearance of a plateau can be seen about 
the sixth day corresponding to cell divisions greater than 2. 
Comparative Example 3A 
Comparative growth tests were carried out for MRC-5 cells coming from the 
28th to 32nd passage on microbeads of the invention, on the one hand, and 
on microbeads of the prior art not coated with reticulated gelatin. 
As microbeads of the prior art, Sephadex G 50 DEAE type microbeads 
(reticulated dextran on which DEAE groups are grafted) were used having a 
charge capacity of the order of 1.5 meq/g of Sephadex G 50. By way of 
comparison, in accordance with the invention, Sephadex G 50 DEAE 
microballs were used coated with reticulated gelatin and prepared as in 
Example 1, having a charge capacity of 1.6 meq/g of dry Sephadex G 50. 
In these comparative tests, the microbeads are subjected to a pretreatment 
such as described in Example 2, in which the microbeads are washed with 
PBS then they are placed in an autoclave, in a PBS medium. 
The seeding step is carried out as in Example 3. Beads and cells are used 
in respective concentrations of 4 mg/ml and 2.5.times.10.sup.5 /ml of 
culture medium. 
A practically zero growth was observed with the microbeads not coated with 
a reticulated protein and more particularly gelatin whereas the number of 
generations obtained with the microbeads of the invention is equal to 2. 
As far as the adhesion rate is concerned, it can be seen that it is of the 
order of 45 in the case of the invention and close to 60 with the 
microbeads of the prior art. 
These results show the advantageous effect procured by the microbeads of 
the invention whose inherent characteristics enable the MRC-5 cells used 
in these tests, which are aged cells which have been subjected to a great 
number of passages, to strongly adhere and to multiply. 
EXAMPLE 4 
Study of the growth of MRC-5 cells having grown on microcarriers of the 
invention 
Several passages of MRC-5 cells were effected over microcarriers of the 
invention formed from type G 50 DEAE Sephadex, covered with gelatin having 
a charge capacity of 1.3 meq/g of Sephadex (before seeding, the 
microcarriers were washed with PBS, then incubated for 30 minutes in PBS 
containing 3% w/v gelatin and finally placed in an autoclave at 
120.degree. C. for 30 minutes in the presence of gelatin). 
The cells were detached then, in 100 ml flask, they were reseeded on their 
old support, the respective concentrations being 2.8.times.10.sup.5 
cells/ml of culture medium for 5 mg/ml of microbeads. 
An adhesion rate of 95% was observed eighteen hours after the subculture of 
the cells and a division number of 2.1. 
Similar tests carried out in 250 to 500 ml flasks, with respectively 
2.5.times.10.sup.5 cells/ml and 1.8.times.10.sup.5 cells/ml for 5 mg/ml of 
microbeads give adhesion rates of 98 and 61 and cellular divisions 
respectively of 2 and 2.47. 
These results show clearly that the cells which have grown on these 
microcarriers of the invention may be reseeded on other carriers, which is 
of great importance for the development of large-scale cultures. 
It will also be noticed that the optimum density obtained in 100 ml 
culture, which is about 10.sup.6 cells/ml, is reproducible in culture 
flasks of larger volume of 250, 500 or 1000 ml, which shows the viability 
of the microcarriers of the invention. 
The whole of the results reported above show that the microbeads of the 
invention offer surfaces particularly suitable for the culture of normal 
diploid cells. As already indicated, these cells, which are on the 
microcarriers, may be infected by a virus or a cellular parasite, which 
allows the elaboration of different cellular products, particularly 
viruses suitable for the manufacture of viral vaccines for human use.