Dried rehydratable film containing agarose or gelose and process for preparing same

Dried agarose or gelose-containing films which are rehydratable into aqueous gel films are disclosed. The dried films are prepared by first forming, on a support, an aqueous gel film containing at most 5% by weight of agarose or gelose and a water-soluble linear polymer or copolymer of acrylamide or methacrylamide. The viscosity of such polymer and copolymer in a 5% aqueous solution at 22.degree. C is about 17000 centipoises or less and preferably about 6000 centipoises or less. The aqueous gel film is then dried according to known techniques, with the linear polymer or copolymer of acrylamide or methacrylamide being included in the dried film.

The present invention has for its object a process of preparing dried films 
containing agarose or gelose, which films can be stored for an extended 
period of time and can be subsequently rehydrated into films of aqueous 
gels of agarose or gelose for use in immunodiffusion, electrophoresis, 
immunoelectrophoresis and immunoelectrodiffusion techniques and processes. 
The invention also relates to the dried films obtained with this process. 
Agarose and gelose are polysaccharides which have been used in the form of 
sheets or films of aqueous gels in the techniques mentioned above. Such 
films contain only a slight amount of agarose or gelose (content less than 
or equal to 5%). The preparation of these films at the time they are to be 
used is troublesome because the user must first dissolve the 
polysaccharide in hot water and then pour the solution obtained onto a 
glass slide. An aqueous gel layer having a thickness of about 2 mm is 
formed upon cooling. Finally the user must make the necessary holes or 
grooves on the surface of the film for the depositing therein of the serum 
or immunizing serum. Furthermore, the aqueous gel sheets produced in this 
manner are fragile, which makes their handling and storage a delicate 
operation. 
For this reason, there has been interest in any process which would make it 
possible to obtain films in a form such that they would be easy to handle 
and to store and which would permit one, by a simple treatment, to convert 
the film into an aqueous gel film of agarose or gelose, ready for use in 
any of the above techniques. 
It has been suggested that the agarose or gelose films should be formed and 
stored in the dehydrated state, but then it is not possible to 
satisfactorily regenerate by rehydration the aqueous gel sheets or films. 
It is known in the art, according to French Pat. No. 1,483,742, how to 
prepare mixed gel films containing, in the aqueous state, 3% to 7% of a 
reticulated polyacrylamide and about 1% agarose or gelose. By 
incorporating glycerin therein at the time of formation of the aqueous 
gel, the resulting dried film would be rehydratable. However, this patent 
does not disclose the manner of dehydration and rehydration nor the length 
of time nor the conditions under which the dehydrated sheets may be 
stored. Furthermore, in the case of simple gel films of agarose or gelose, 
the incorporation of glycerin at the time of formation of the aqueous gel 
does not make it possible to obtain films which can be rehydrated after 
dehydration. 
The present invention has for it object a solution to the problem of 
forming, from aqueous gel film containing at most 5% by weight agarose or 
gelose, a dried film that is easy to handle and store and at the same time 
is totally rehydratable even after an extended period of storage time, to 
again form an aqueous gel film of agarose or gelose, ready for use in any 
of the aforementioned techniques or processes. 
According to the present invention, it has been found that such 
rehydratable films or sheets can be formed if there is incorporated in the 
agarose or gelose-containing aqueous gel, prior to drying by any known 
process, a water-soluble linear polymer or copolymer of acrylamide or 
methacrylamide, whose viscosity in a 5% aqueous solution at a temperature 
of 22.degree. C, is 17000 centipoises or less, and preferably, 6000 
centipoises or less. 
As water-soluble linear copolymers of acrylamide or methacrylamide that can 
be used for the purpose of this invention, there can be cited, for 
example, linear copolymers of acrylamide or methacrylamide with 
vinylpyrrolidone and linear copolymers of acrylamide or methacrylamide 
with an acrylic or methacrylic ester, such as monomethacrylate or the 
acrylate of ethylene glycol. The percentage by weight of acrylamide or 
methacrylamide of the linear copolymers is, preferably, at least equal to 
30%. 
The process according to the invention consists in preparing a hot 
glycero-aqueous solution containing by weight 2-3% glycerol. The solution 
also has therein the desired amounts of agarose (or gelose) and of linear 
polymer or copolymer of acrylamide or methacrylamide. The solution, which 
is preferably at a temperature of 75.degree. C to 80.degree. C, is poured 
onto a support, to form a layer or film of desired thickness, such as for 
example, 2mm, and the solution is then cooled. The aqueous gel thus formed 
is covered with a sheet of regenerated cellulose, such as cellophane, 
which has been wetted with an aqueous solution of glycerol, preferably a 
2% to 3% glycerol solution or wetted with an aqueous solution of glycerol 
and acetic acid, preferably a 2% to 3% glycerol and 3% acetic acid 
solution. Finally the film is dried at a temperature of less than 
30.degree. C, preferably by passing a current of air thereover. Thus, 
there is formed on the support and covered with a sheet of cellophane, a 
flexible dried film containing the agarose or gelose and the linear 
polymer or copolymer of acrylamide or methacrylamide. The glycerol imparts 
flexibility to the film, and the film has a 3% to 7% moisture content. 
This film is then stored in an impermeable plastic container or bag and is 
maintained, preferably, at a temperature of about 4.degree. C. 
The dried films thus prepared and stored have a long, extended preservation 
time of at least 8 months when stored at 4.degree. C and a shelf life of 
at least 3 months when stored at 20.degree. C. Furthermore, they are easy 
to use in any of the aforesaid techniques. All that is required is to 
immerse the film for several hours in a buffer solution or preferably in 
renewed (renouvellee) water, at ambient temperature, and the film is then 
completely rehydrated to form an aqueous gel film of agarose or gelose 
ready for immediate use. At the time of rehydration, the linear polymer or 
copolymer of acrylamide or methacrylamide, being soluble in water, is 
removed from the gel, so that there remains the aqueous gel film of 
agarose or gelose. Furthermore, at the time of rehydration, the cellophane 
sheet is detached from the film, but this latter adheres to the support 
which makes its further handling easy. Buffers that can be used for the 
rehydration process include, for example, barbital (0.005 M, pH 8.4), tris 
(hydroxymethyl)aminomethane ( 0.05 M adhjusted to pH 7.4 by HC1), and 
tris(hydroxymethyl)aminomethane ( 0.01 M adjusted to pH 8.7 by glycocoll). 
Furthermore, before covering the aqueous gel with the cellophane sheet and 
then drying it, it is possible to make whatever holes, grooves, and the 
like, in the gel surface which may be necessary for subsequent use of the 
film, after rehydration, in the techniques of immunodiffusion, 
electrophoresis, immunoelectrophoresis and immunoelectrodiffusion 
The concentrations of agarose (or gelose) and of linear polymer or 
copolymer of acrylamide or methacrylamide of the glycero-aqueous solution 
used in the process according to the invention, and consequently the 
concentrations of these constitutents in the aqueous gel obtained before 
drying, are preferably between 0.6% and 1.5% by weight of agarose (or 
gelose) and between 3% and 6% by weight for the linear polymer or 
copolymer of acrylamide or methacrylamide. 
The linear polymers or copolymers of acrylamide or methacrylamide used in 
the process according to the invention can be prepared, according to known 
methods, by polymerization or copolymerization in an aqueous medium of the 
monomer or monomers, in the presence of catalyst systems such as N,N,N',N' 
tetramethyl ethylene diamine (TEMED) and an alkaline persulfate, 
dimethylaminopropionitrile and an alkaline persulfate, riboflavin and an 
alkaline persulfate in the presence of ultra-violet (UV) rays, and 2,2' 
-azo-bis-isobutyronitrile. The polymer or copolymer formed is then 
separated by dialysis and/or precipitation in an alcohol medium, 
dehydrated and then dried. 
The linear polymers or copolymers of acrylamide or methacrylamide used in 
the process according to the invention can also be prepared by an original 
method which offers the advantage, over the standard method described 
above, of providing linear polymers or copolymers whose aqueous or 
glycero-aqueous solutions are often clearly less viscous than those of the 
linear polymers or copolymers obtained by the standard methods, which 
facilitates the use of the products at the time of preparation of the 
aqueous gel sheets. According to this original method the polymerization 
or copolymerization of the monomer or monomers is performed, in the 
presence of one of the catalyst systems previously mentioned, but, instead 
of a pure aqueous medium, there is used an aqueous medium containing a 
water-soluble non-copolymerizable organic compound such as, for example, a 
polyol, a sugar, a polysaccharide or a polyvinylpyrrolidone. Examples of 
polyols that can be used include, in particular, glycols, glycerol, 
sorbitol and polyethylene glycols having an average molecular weight of 
200 to 600. Examples of polyvinylpyrrolidones that can be used include, in 
particular, polyvinylpyrrolidones having an average molecular weight of 
10,000 to 40,000. The weight of the water-soluble noncopolymerizable 
organic compound used is preferably equal to or approximate to the weight 
of the monomer or monomers used. The linear polymers or copolymers of 
acrylamide or methacrylamide thus formed are then separated, in a form 
free of the above organic compound, by dialysis and/or precipitation in an 
alcohol medium, dehydrated and dried. 
As supports that can be used in the preparation of the dried sheets by the 
process according to the invention there can be cited, in particular, the 
transparent plastic supports for photographic films, based on treated 
polyester, known on the market by the name "Cronar Clear Base" and sold by 
E. I. Du Pont de Nemours and Co., Wilmington, Del. 19898, USA. It has been 
found that these supports possess the required adherence qualities with 
respect to the films of aqueous gel of agarose or gelose, contrary to 
other supports, such as cellulose triacetate films or ethylene glycol 
polyterephthalate films, known by the trademark .-+.Mylar.

The following examples, while designed to illustrate the invention, are in 
no way to be considered as limiting the scope of the invention thereto: 
EXAMPLE 1 
1. Preparation of the linear acrylamide polymer 
Into one liter of demineralized water at ambient temperature there are 
dissolved 100 g of pure crystallized acrylamide monomer and 0.2 ml of 
N,N,N',N' tetramethyl ethylene diamine (TEMED). The pH of the solution is 
adjusted to 6.8- 7 with HCl, and then 0.8 g of ammonium persulfate is 
added. The solution is thereafter heated for 2 hours at 50.degree. C. 
After cooling, the solution is first dialyzed and then the polymer is 
precipitated by addition of 4 volumes of methanol to the dialysate and 
stirring the mixture. The precipitate obtained is separated by decanting, 
then taken up again by methanol and dehydrated by grinding in this 
solvent. The polymer is then drained and dried under vacuum at 60.degree. 
C. 95 to 100 g of linear acrylamide polymer are produced, in the form of a 
white water-soluble powder, whose viscosity, in a 5% aqueous solution at 
22.degree. C, is equal to 17000 centipoises. 
2. Preparation of dried agarose (or gelose) films 
2 g of agarose (or gelose) are dissolved in 100 ml of boiling demineralized 
water containing 2% glycerol in a boiling water bath 8 g of the linear 
acrylamide polymer prepared as indicated above are dissolved in 100 ml of 
demineralized water containing 2% glycerol at ambient temperature. The two 
solutions are brought to 75.degree. - 80.degree. C and then mixed while 
slowly stirring to avoid the formation of air bubbles. The mixture, while 
at this same temperature, is then poured onto a transparent plastic 
support, "Cronar Clear Base C-72", which has been placed on a horizontal 
glass slide, and a gel layer 2 mm thick is formed. The layer is cooled to 
a temperature below 25.degree. C and forms an aqueous gel that adheres to 
the plastic support. Holes and grooves are formed on the surface of the 
gel by means of an appropriate tool, and the gel that fills the holes is 
removed by aspiration with a Pasteur pipette. The aqueous gel film is then 
covered with a cellophane sheet previously wetted in a glycero-aqueous 
solution containing 2% glycerol. The edges of the cellophane sheet are 
folded under the glass slide and the unit is dried at ambient temperature 
in an air current over night. 
The unit, which consists of the dried gel film, the "Cronar Clear Base 
C-72" support and the cellophane sheet is then detached from the glass 
slide and stored under the conditions indicated above. The dried film, 
thus obtained and stored, has a long storage life and is totally 
rehydratable. 
EXAMPLE 2 
1. Preparation of the linear acrylamide polymer 
Into a liter of demineralized water at ambient temperature there are 
dissolved 100 g of pure crystallized acrylamide monomer, 100 g of powdered 
polyvinylpyrrolidone with a molecular weight of 11000 and 1 ml of TEMED. 
The pH of the solution is adjusted to 6.8-7 with N HCL and then 0.8 g of 
ammonium persulfate is added thereto. The solution thus formed is heated 
for 5 hours at 50.degree. C and then allowed to cool to ambient 
temperature. The solution is then dialyzed with reference to demineralized 
water, and the polymer is precipitated by addition of 5 volumes of 
methanol to the dialysate and vigorously stirring the mixture. The 
precipitate obtained is separated by decanting, then taken up by methanol 
and dehydrated by grinding in solvent. The polymer is then drained and 
dried under vacuum at 60.degree. C. 90 to 95 g of linear acrylamide 
polymer are produced, in the form of a white water-soluble powder, whose 
viscosity in 5% aqueous solution at 22.degree. C, is less than 6000 
centiposes. 
2. Preparation of dried agarose (or gelose) films 
The process of Example 1 is repeated except that the 8 g of linear 
acrylamide polymer of Example 1 are replaced by 12 g of the linear 
acrylamide polymer formed as described above by polymerization in the 
presence of polyvinylpyrrolidone having a molecular weight of 11,000. 
The dried film obtained has a long storage life and is totally 
rehydratable. 
EXAMPLE 3 
1. Preparation of linear acrylamide polymer 
The process of Example 2 is repeated except that the 100 g of 
polyvinylpyrrolidone having a molecular weight of 11,000 are replaced with 
100 g of polyethyleneglycol having a molecular weight of 600 and 2 ml of 
TEMED are used instead of 1 ml. 90 to 95 g of linear acrylamide polymer 
are produced, in the form of a white water-soluble powder. Its viscosity, 
in 5% aqueous solution at 22.degree. C, is less than 6000 centipoises. 
2. Preparation of dried agarose (or gelose) films 
The process of Example 1 is repeated except that the 8 g of linear 
acrylamide polymer of Example 1 are replaced by 12 g of the linear 
acrylamide polymer produced above by polymerization in the presence of 
polyethylene glycol having a molecular weight of 600. 
The dried film obtained has a long storage life and is totally 
rehydratable. 
EXAMPLE 4 
1. Preparation of linear acrylamide polymer 
The process of Example 2 is repeated except that the 100 g of 
polyvinylpyrrolidone having a molecular weight of 11,000 are replaced by 
100 g of glycerol. 90 to 95 g of linear acrylamide polymer are produced, 
in the form of a white water-soluble powder, whose viscosity in 5% aqueous 
solution at 22.degree. C, is less than 6000 centipoises. 
2. Preparation of dried agarose (or gelose) films 
The process of Example 1 is repeated except that the 8 g of linear 
acrylamide polymer of Example 1 are replaced by 12 g of linear acrylamide 
polymer as formed above by polymerization in the presence of glycerol. 
The dried film which is produced has a long preservation time and is 
totally rehydratable. 
EXAMPLE 5 
1. Preparation of linear acrylamide polymer 
The process of Example 2 is repeated except that the 100 g of 
polyvinylpyrrolidone having a molecular weight of 11,000 are replaced by 
100 g of sorbitol. 90 to 95 g of linear acrylamide polymer are produced, 
in the form of a white water-soluble powder, whose viscosity in 5% aqueous 
solution at 22.degree. C, is less than 17000 centipoises. 
2. Preparation of dried agarose (or gelose) sheets 
The operation of Example 1 is repeated except that the 8 g of linear 
acrylamide polymer of Example 1 are replaced by 6 g of linear acrylamide 
polymer as produced above by polymerization in the presence of sorbitol. 
The dried film obtained has a long preservation time and is totally 
rehydratable. 
EXAMPLE 6 
1. Preparation of the linear acrylamide copolymer 
To 900 ml of demineralized water at ambient temperature are added 50 g of 
pure crystallized acrylamide monomer and 50 ml of vinylpyrrolidone. The 
solution is stirred for 15 minutes and is then filtered. The filtered 
solution is brought to 50.degree. C and 2 ml of TEMED are added thereto. 
The pH is adjusted to 6.8-7 with N HCl and then 1 g of ammonium persulfate 
is dissolved in the solution. The solution is kept at 50.degree. C for 5 
hours and is then cooled to ambient temperature. The solution is then 
dialyzed with reference to demineralized water, and the copolymer is 
precipitated by the addition of 10 volumes of methanol to the dialysate, 
while the mixture is stirred. The precipitate which forms is separated by 
decanting, then taken up by methanol and dehydrated by grinding in this 
solvent. The copolymer is then drained and dried under vacuum at 
60.degree. C. A linear copolymer of acrylamide and vinylpyrrolidone is 
produced in the form of a water-soluble white powder, whose viscosity in 
5% aqueous solution at 22.degree. C, is less than 6000 centipoises. 
2. Preparation of dried agarose (or gelose) films 
The process of Example 1 is repeated except that the 8 g of the linear 
acrylamide polymer of Example 1 are replaced by 12 g of acrylamide and 
vinylpyrrolidone linear copolymer produced above. 
The dried film obtained has a long storage life and is totally 
rehydratable. 
EXAMPLE 7 
1. Preparation of linear acrylamide copolymer 
The process of Example 6 is repeated except that at the start there are 
added 100 ml of polyethylene glycol with a molecular weight of 600. A 
linear copolymer of acrylamide and vinylpyrrolidone is obtained, in the 
form of white water-soluble powder, whose viscosity in 5% aqueous solution 
at 22.degree. C, is less than 6000 centipoises. 
2. Preparation of the dried agarose (or gelose) films 
From the linear copolymer of acrylamide and vinylpyrrolidione produced 
above by copolymerization in the presence of the polyethylene glycol of a 
molecular weight of 600, there are prepared, by utilizing the process of 
Example 6 , dried agarose (or gelose) films. 
The dried films obtained have a long storage life and are totally 
rehydratable.