Diagnostic test for virus desease and method for the cryopreservation of cells used in these tests.

p0001The present invention relates to a diagnostic test for viral disease and a useful cell-freezing method for storing cells for said diagnostic test.

p0002The diagnosis of infectious diseases and the identification of the agent required to present a quick and accurate response to enable the treatment of the disease with the appropriate drug or the decision of a particular medical procedure. In the case of viral diseases, early diagnosis is made necessary for vis-à-vis which treatment is currently possible virus (Herpes simplex virus or HSV) or where accurate identification is important (cystomegalovirus, syncytiel respiratory viruses .. .).

p0003The laboratory diagnosis of virus is based on two methods on one hand, the direct detection of specific antigens, on the other hand, culture of the virus on cells susceptible strains. The first method involves the use of antibodies (monoclonal or polyclonal) antibodies recognizing one or more viral antigens characteristic of the species, and detected by immunofluorescence, immunoperoxidase, and ELISA more recently probe to DNA or RNA. The advantage of this method is the specific identification of the virus (antigen) of live virus particles is not required. The major disadvantages of this method are the lack of sensitivity, the need for good quality sample and technical variables to distinguish a positive specimen of a negative sample (Schmidt<u>stall</u>, 1983, J. Clin. Microbiol. 18: 445-448; Nerurkar<u>stall</u>, 1984, J. Clin.Microbiol,. 19: 631: 633).

p0004The second method of virus diagnostic uses of cell strains maintained continuously under laboratory conditions. These cells can amplify the virus to identify, from samples, and make its detection possible after 24 hours by observing cytolysis range, identification of viral antigens with specific antibodies or the use of probe DNA. This method remains the gold standard for its lively sensitivity and amplification equipment necessary for an accurate diagnosis.

p0005However the culture of cells continuously required by these techniques of diagnosis constitutes a major drawback in their use.

p0006Therefore the present invention provides a method of freeze-thaw cells allowing their use for viral diagnosis within 24 hours.

p0007The present invention therefore provides a diagnostic test for viral disease comprising the use of cells to amplify the virus to be identified, from samples and making its detection possible after culture, characterized in that the cells the frozen thawed for testing.

p0008According to the diagnostic tests according to the invention the detection of the virus can be done by iden tification of viral antigens by specific antibodies, the use of DNA probes or simply observing cytolysis range.

p0009There may be mentioned for illustrative and not limiting the tests to detect herpes simplex virus, cytomegalovirus, or respiratory syncytiel virus.

p0010According to the invention the infective virus sample can be mixed with the cells immediately after thawing, the viral agent that can be identified after incubation for 24 hours by a specific method (antigen, DNA).

p0011The present invention therefore also relates to a cell-freezing process in a particularly consistent base container in multiwell plates or tubes allowing the use of cells after thawing, in particular in a diagnostic test according to the invention. The development of freeze-thaw methods according to the invention presents some challenges to the extent that it is necessary to maintain viability after thawing a further optimal cells, also a sensitivity to the viral agent.

p0012According to the essential feature of this process, - Firstly using a cryopréservant chosen so that the number of viable and functional cells, that is to say keeping sensitivity to the viral agent, after thawing is optimal and especially greater than 80%. - Secondly, the number of cells to be frozen inserted into the support container is selected so that the cultured cells form a complete monolayer 24 hours after thawing.

p0013So are satisfied the best conditions to perform the viral diagnostic test, particularly when detection is done by observing cytolysis range.

p0014Advantageously, the carrier container is of plastics material previously treated with a coating agent selected in particular from collagen, fibronectin, polylysine, concanavalin and mixtures thereof, in particular the supports are treated with a mixture of collagen and fibronectin.

p0015The supports thus treated it possible to obtain: -on the one hand good flow, that is to say a uniform cell spreading and -on the other hand a better attachment of the cells on the support, which are also the ideal conditions to perform the diagnostic test.

p0016The cryopréservant agent according to the invention consists of a cryoprotectant diluted.

p0017There may be mentioned as cryoprotective agent according to the invention DMSO, glycerol, trehalose, dimethylacetamide, polyéthylèneglycérol, the polyvinyl pyrolidone.

p0018The diluent may be the only culture medium, preferably the medium supplemented with fetal calf serum (FCS) in particular 5 to 10%, or bovine serum albumin in particular 2 to 5%, or the pure SFV.

p0019Can perform the freezing of cells suspended in a container being the container carrier or in monolayer culture in the support container.

p0020Include cells as an illustration: . embryonic fibroblast cell type of lung human MRC-5, and . monkey kidney fibroblast cell type VERO.

p0021The use of the freezing process according to the present invention is particularly useful for the amplification of viruses for their identification, but such use is not limiting.

p0022Other advantages and features of the invention appear in the light of the following examples.

example 1

:

CULTURE CELLS

p0023The cell line MRC-5 (embryonic fibroblast-like human lung) and VERO line (monkey kidney fibroblast cell type) were chosen as examples. These two lines are susceptible to herpes virus, cytomegalovirus and respiratory syncytiel virus and are currently the most used. Other cell lines may be considered for the development of other viral strains.

p0024The cells are cultured at 37 ° C in an atmosphere saturated with water in the ratio CO₂ / air (7:93, v / v) in tissue culture flasks in plastic 175 cm² (T175). The medium consists of RPMI-1640 containing 10 mM NaHCO₃, 20 mM Hepes, 2 mM glutamine, 50μg / ml streptomycin, 50 U / ml penicillin and supplemented with 1% of a solution of amino acids not essential and 10% fetal calf serum inactivated by heat (SFV). The medium should be repeated 2 times per week. The cultures are transplanted from cell confluence every 5-7 days depending on the cell type. The passaged cultures are prepared by trypsinization of the monolayers with a trypsin-EDTA mixture comprising 0.05% (w / v) trypsin and 0.02% (w / v) EDTA. To do this, the old medium is removed, cells washed with buffered saline and incubated minutes at 37 ° C with trypsin-EDTA solution. The cells are resuspended in culture medium and transferred into new dishes with subculturing ratio of 1: 5.

example 2

:

PARAMETERS EVALUATION AND SENSITIVITY TO VIRAL INFECTION.

p00251) Various criteria were examined to evaluate the culture cells after the process of freezing and thawing. The parameters used are as follows:

p0026<u>Cell viability</u>After thawing, is determined by the trypan blue exclusion test (0.5% solution by weight in a phosphate buffer saline solution) and the test of the tetrazolium salt (MTT) according to the described by technical and Gerlier Thomasset (. J. Immunol Methods 1986, 94: 57-63). This test assesses cell viability based on the biochemical function of the cells. The compound, colorless and soluble in the culture medium, is converted by enzymes mitochondrieux (dehydrogenase) in crystals of colored and insoluble formazan. The precipitate is dissolved in an isopropanol solution containing 0.04 N HCl and its concentration is determined using a spectrophotometer at wavelengths of 570 nm and 630 nm. Automatic reading can be performed in an ELISA reader to multiwell plate. A linear regression of the concentration of the processed product and the number of cells is established for each cell line and allows to estimate the number of viable cells (functional) of each of the preparations studied.

p0027<u>The number of total cells</u> is determined either by counting the cells in a hemocytometer after staining nuclei undiluted Turck blue, or after fixation of the cells in culture and staining of the nuclei with haematoxylin. To do this, the cells are fixed with a glacial acetic acid-methanol solution (1: 3, v: v) at a volume of fixative per volume of culture medium. After 5 minutes, the medium is aspirated and cells are washed 2x 2 minutes with the same fixative. After rinsing with water to remove the remaining fixative, the cells were air dried and then stained with haematoxylin (commercial solution) for 5 minutes. The cells are then rinsed with tap water 2 to 3 times and then dried again.

p0028This test allows for automatic reading using an ELISA reader at wavelength of 570 nm. A linear regression is also established between the measured absorbance and the total number of cells, and this, for each of the two cell lines.

p0029<u>Microscopic observation</u> phase contrast is used to define the morphological appearance of the cells, cell spreading and distribution of the cells on the support (homogeneous or not), as well as the appearance of the cell monolayer at confluency of the cells. The time required to obtain a perfect confluent cell layer is also determined by microscopic observation.

p00302) The methods provided by the invention are intended to allow the preservation of cells, used for the diagnosis of viral disease, to enable the appropriate time to carry out the diagnostic test.

p0031It is therefore important to determine whether the cells having undergone the freezing process + thawing retained sensitivity to the viral agent and to what degree.

p0032We considered Herpes simplex virus type I (HSV-1) and type II (HSV-2). Both virus strains obtained from the ATCC under code number VR-733 (HSV-1) and VR-734 (HSV-2). Viruses are grown on HEp 2 cells in MEM supplemented with 10% FCS and stored at -70 ° C at 2,3.10⁸ 7.10⁷ and viral particles per ml of complete culture medium, respectively for the HSV- 1 and HSV-2.

p0033A diagnostic test is to infect the cells (MRC-5 and Vero cells) confluent in 24-well plates with increasing dilution (10 X 10 X) of the viral inoculum, the culture medium. To this end, the culture medium is aspirated and replaced with the viral suspension at a rate of 100 .mu.l per well (minimum volume covering the cells without further drying). The cells are incubated at 37 ° C for 1 hour. Fresh medium is then added to each well and the cells are incubated at 37 ° C. The formation of cytolysis ranges, characteristics for the two strains of Herpes are observed in phase contrast microscopy after 24 and 48 hours.

p0034This test is performed for the cells in culture and the cells frozen and thawed according to the methods developed below.

example 3

:

FREEZING IN CELL CULTURE MONOLAYER

3.1 Culture conditions

p0035Cells MRC-5 and Vero cells, grown in the culture medium described above, are placed in 24 well plates (Linbro, Flow Laboratories) or Gencell tubes (GENUS DIAGNOSTICS) in an amount of 5x10⁴ cells per well or tube. The cells are maintained at 37 ° until a complete monolayer (after 24 to 48 hours).

3.2 Method of freezing and thawing

p0036The various factors mentioned above, have been studied for each cell types and conditions.

p0037The best conditions defined for the freeze-thaw MRC-5 cells in monolayer culture in Gencell tubes are as follows. At confluence the cells, the cryopréservant, a 10% solution of trehalose in the fetal calf serum is applied to the cells. The tube is maintained 10 minutes at 20 ° before being frozen directly at -70 ° (-5 ° C / min). The tubes are stored at this temperature. A thawing, the tube is maintained at 20 ° until dissolution of cryopréservant. Thereof is gently removed and replaced with complete culture medium at room temperature. The cells are maintained at this temperature for 10 to 15 minutes before being incubated at 37 ° with 7% CO₂ in air. The culture medium was renewed after 24 hours.

p0038The same procedure is applicable to MRC-5 cells and in Vero cells grown in 24-well plates, the cryopréservant being, in the case of Vero cells, DMSO in 7.5% FCS.

p0039The cell media are processed before culture with collagen and fibronectin. This coverage is obtained by incubating the boxes or tubes for 24 hours at 37 ° with 1 ml of collagen (100 ug / ml) and fibronectin (50 micrograms / ml) in phosphate buffer solution and washing subsequently with the same buffer. The supports are used as such or are dried prior to 20 °.

3.3 Results

p0040The effect of 3 cryopréservants on cell viability and cell attachment MRC-5 Gencell tube support is shown in Table 1. The 7.5% DMSO and 5% glycerol in SFV do not allow to maintain the cells attached to the support. Glycerol 10% keeps 37% of the cells attached to the support after 24 hours, but the cellular distribution is heterogeneous and slower growth than cells in normal culture. The 10% trehalose in FCS gives the best results with 62% of attached cells after 24 hours and good viability. The cells are distributed evenly and faster growing.

p0041In Table 2, it appears that 75% (± 13%) of the available area (tube Gencell) is occupied by the MRC-5 cells, 24 hours after thawing when the 10% trehalose in FCS is used as cryopréservant. After 48 hours, the cells occupy only 45% (± 34%) of the surface. The results show a pronounced cell detachment between 24 and 48 hours and significant variation is observed series to series. Cells multiply 48 hours after thawing and require 6-9 days prior to reform a confluent cell and can not be used before 48 hours.

p0042To improve cell attachment, <u>the cells are grown on treated surfaces</u> collagen (100 micrograms) and fibronectin (100 mcg). Polylysine and concanavalin A prevent the spreading and growth of cells.

p0043Under these conditions (Table 2), the cell detachment after 48 hours is significantly reduced with greater homogeneity in each series. Cell growth is faster, reaching confluence in 3-5 days. The cells are useful for diagnostic tests after 48 hours of culture.

p0044It should be noted that the treatment of the support, further improves the effect of 10% glycerol in SFV.

p0045Currently, the surfaces are treated with a mixture of collagen and fibronectin in the respective concentration of 100 micrograms and 50 micrograms.

p0046When freezing of cells in monolayer culture in 24 well plates, similar results are obtained thawing (Table 3). <tables id="tabl0001" num="0001"><table frame="all"><title>Table 1</title><tgroup cols="7" colsep="1" rowsep="0"><colspec colnum="1" colname="col1" colwidth="22.50mm" /><colspec colnum="2" colname="col2" colwidth="22.50mm" /><colspec colnum="3" colname="col3" colwidth="22.50mm" /><colspec colnum="4" colname="col4" colwidth="22.50mm" /><colspec colnum="5" colname="col5" colwidth="22.50mm" /><colspec colnum="6" colname="col6" colwidth="22.50mm" /><colspec colnum="7" colname="col7" colwidth="22.50mm" /><thead valign="top"><row><entry namest="col1" nameend="col7" align="center">Study the viability of MRC-5 cells in monolayer and cell attachment after thawing according cryopréservants.</entry></row><row><entry namest="col1" nameend="col2" align="center">Cryopréserv%. in SFV</entry><entry namest="col3" nameend="col4" align="center">Viability (%)^at</entry><entry namest="col5" nameend="col5" align="center">Cell layer.^b (24 h)</entry><entry namest="col6" nameend="col6" align="center">cell division</entry><entry namest="col7" nameend="col7" align="center">cell growth</entry></row><row><entry namest="col1" nameend="col1" /><entry namest="col2" nameend="col2" /><entry namest="col3" nameend="col3" align="center">2 h</entry><entry namest="col4" nameend="col4" align="center">24</entry><entry namest="col5" nameend="col5" /><entry namest="col6" nameend="col6" /><entry namest="col7" nameend="col7" /></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left">DMSO</entry><entry namest="col2" nameend="col2" align="right">7.5%</entry><entry namest="col3" nameend="col3" align="right">50</entry><entry namest="col4" nameend="col4" align="right">-</entry><entry namest="col5" nameend="col5" align="right">detachment</entry><entry namest="col6" nameend="col6" align="left">-</entry><entry namest="col7" nameend="col7" align="left">-</entry></row><row><entry namest="col1" nameend="col1" align="left">Glycerol</entry><entry namest="col2" nameend="col2" align="right">5%</entry><entry namest="col3" nameend="col3" align="right">50</entry><entry namest="col4" nameend="col4" align="right">-</entry><entry namest="col5" nameend="col5" align="right">detachment</entry><entry namest="col6" nameend="col6" align="left">-</entry><entry namest="col7" nameend="col7" align="left">-</entry></row><row><entry namest="col1" nameend="col1" /><entry namest="col2" nameend="col2" align="right">10%</entry><entry namest="col3" nameend="col3" align="right">70</entry><entry namest="col4" nameend="col4" align="right">80</entry><entry namest="col5" nameend="col5" align="right">37% (30)</entry><entry namest="col6" nameend="col6" align="left">heterogeneous</entry><entry namest="col7" nameend="col7" align="left">slow</entry></row><row><entry namest="col1" nameend="col1" /><entry namest="col2" nameend="col2" align="right">15%</entry><entry namest="col3" nameend="col3" align="right">50</entry><entry namest="col4" nameend="col4" align="right">80</entry><entry namest="col5" nameend="col5" align="right">13% (24)</entry><entry namest="col6" nameend="col6" align="left">heterogeneous</entry><entry namest="col7" nameend="col7" align="left">slow</entry></row><row><entry namest="col1" nameend="col1" align="left">trehalose</entry><entry namest="col2" nameend="col2" align="right">5%</entry><entry namest="col3" nameend="col3" align="right">70</entry><entry namest="col4" nameend="col4" align="right">75</entry><entry namest="col5" nameend="col5" align="right">50% (20)</entry><entry namest="col6" nameend="col6" align="left">homogeneous ±</entry><entry namest="col7" nameend="col7" align="left">normal ±</entry></row><row><entry namest="col1" nameend="col1" /><entry namest="col2" nameend="col2" align="right">10%</entry><entry namest="col3" nameend="col3" align="right">75</entry><entry namest="col4" nameend="col4" align="right">85</entry><entry namest="col5" nameend="col5" align="right">62% (27)</entry><entry namest="col6" nameend="col6" align="left">homogeneous ±</entry><entry namest="col7" nameend="col7" align="left">normal ±</entry></row></tbody></tgroup><tgroup cols="7" colsep="0" rowsep="0"><colspec colnum="1" colname="col1" colwidth="22.50mm" /><colspec colnum="2" colname="col2" colwidth="22.50mm" /><colspec colnum="3" colname="col3" colwidth="22.50mm" /><colspec colnum="4" colname="col4" colwidth="22.50mm" /><colspec colnum="5" colname="col5" colwidth="22.50mm" /><colspec colnum="6" colname="col6" colwidth="22.50mm" /><colspec colnum="7" colname="col7" colwidth="22.50mm" /><tbody valign="top"><row><entry namest="col1" nameend="col7" align="justify">^at Cell viability was evaluated 2 and 24 hours after thawing and is expressed in% of the number of cells present at 2 h and 24 h in Gencell tubes.</entry></row><row><entry namest="col1" nameend="col7" align="justify">^b Area occupied by cells 24 h after thawing in% of the total area (average standard ± n = 4 sets corresponding to 14 tubes) and calculated from the total number of cells present at 24 h and at confluence (control cultures) .</entry></row></tbody></tgroup></table></tables><tables id="tabl0002" num="0002"><table frame="all"><title>Table 2</title><tgroup cols="4" colsep="1" rowsep="0"><colspec colnum="1" colname="col1" colwidth="39.37mm" /><colspec colnum="2" colname="col2" colwidth="39.37mm" /><colspec colnum="3" colname="col3" colwidth="39.37mm" /><colspec colnum="4" colname="col4" colwidth="39.37mm" /><thead valign="top"><row><entry namest="col1" nameend="col4" align="center">tube support processing Gencell Influence on cell attachment after thawing MRC-5 cells in monolayer.</entry></row><row><entry namest="col1" nameend="col4" align="center">Freezing of support "Treaty"</entry></row><row><entry namest="col1" nameend="col1" rowsep="0" align="center">Cryopréservant</entry><entry namest="col2" nameend="col4" align="center">Support^at</entry></row><row><entry namest="col1" nameend="col1" /><entry namest="col2" nameend="col2" rowsep="0" align="center">Control</entry><entry namest="col3" nameend="col3" rowsep="0" align="center">collagen</entry><entry namest="col4" nameend="col4" rowsep="0" align="center">fibronectin</entry></row><row><entry namest="col1" nameend="col1" align="left"><u>10% trehalose</u></entry><entry namest="col2" nameend="col2" /><entry namest="col3" nameend="col3" /><entry namest="col4" nameend="col4" /></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left">24</entry><entry namest="col2" nameend="col2" align="right">75% (13)</entry><entry namest="col3" nameend="col3" align="right">94% (9)</entry><entry namest="col4" nameend="col4" align="right">77% (8)</entry></row><row><entry namest="col1" nameend="col1" align="left">48 h</entry><entry namest="col2" nameend="col2" align="right">45% (34)</entry><entry namest="col3" nameend="col3" align="right">88% (13)</entry><entry namest="col4" nameend="col4" align="right">75% (5)</entry></row><row><entry namest="col1" nameend="col1" align="left">confluence^b</entry><entry namest="col2" nameend="col2" align="right">6-9</entry><entry namest="col3" nameend="col3" align="right">3-5</entry><entry namest="col4" nameend="col4" align="right">4-6</entry></row></tbody></tgroup><tgroup cols="4" colsep="1" rowsep="0"><colspec colnum="1" colname="col1" colwidth="39.37mm" /><colspec colnum="2" colname="col2" colwidth="39.37mm" /><colspec colnum="3" colname="col3" colwidth="39.37mm" /><colspec colnum="4" colname="col4" colwidth="39.37mm" /><thead valign="top"><row><entry namest="col1" nameend="col1" align="left"><u>10% glycerol</u></entry><entry namest="col2" nameend="col2" /><entry namest="col3" nameend="col3" /><entry namest="col4" nameend="col4" /></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left">24</entry><entry namest="col2" nameend="col2" align="right">62% (14)</entry><entry namest="col3" nameend="col3" align="right">90% (8)</entry><entry namest="col4" nameend="col4" align="right">68% (9)</entry></row><row><entry namest="col1" nameend="col1" align="left">48 h</entry><entry namest="col2" nameend="col2" align="right">20% (0)</entry><entry namest="col3" nameend="col3" align="right">75% (17)</entry><entry namest="col4" nameend="col4" align="right">66% (4)</entry></row><row><entry namest="col1" nameend="col1" align="left">confluence^b</entry><entry namest="col2" nameend="col2" align="right">11</entry><entry namest="col3" nameend="col3" align="right">3-6</entry><entry namest="col4" nameend="col4" align="right">4-7</entry></row></tbody></tgroup><tgroup cols="4" colsep="0" rowsep="0"><colspec colnum="1" colname="col1" colwidth="39.37mm" /><colspec colnum="2" colname="col2" colwidth="39.37mm" /><colspec colnum="3" colname="col3" colwidth="39.37mm" /><colspec colnum="4" colname="col4" colwidth="39.37mm" /><tbody valign="top"><row><entry namest="col1" nameend="col4" align="justify">^at The results represent the area occupied by cells 24 and 48 hours after thawing (n = 2 sets corresponding to 8 tubes).</entry></row><row><entry namest="col1" nameend="col4" align="justify">^b Represents the number of days required to obtain a monolayer of confluent cells.</entry></row></tbody></tgroup></table></tables>

example 4

:

FREEZING CELLS SUSPENDED PLATE multiwell

4.1 Methods

p0047Cells MRC-5 and Vero cells were detached from the culture dishes using trypsin-EDTA solution. After detachment, the cells were resuspended in complete medium, centrifuged and resuspended in the same medium. After counting, the cells were centrifuged again and then resuspended in the cryopréservant, a 7.5% solution of DMSO in FCS at a final cell concentration of 2,4x10⁵ MRC-5 cells / ml and 4,4x10⁵ Vero cells / ml.

p0048Suspension cells are distributed into 24-well plates (Linbro, Flow Laboratory) at 0.5 ml / well and then frozen directly at -70 ° C. The wells of each plate were pre-treated with a mixture of collagen and fibronectin as described in Example 1.2.

p0049A thawing, the plates are placed at 37 ° until complete solubilization of cryopréservant. A volume of 1.5 ml of serum-free fresh medium is added to each well, and the plate is centrifuged for 5 minutes at 1500 rpm. The medium is then replaced with complete medium. The cells are resuspended and centrifuged again for 5 minutes. The cells are then incubated at 37 ° C under 7% CO₂ in air. The medium is renewed after 24 hours.<tables id="tabl0003" num="0003"><table frame="all"><title>Table 3</title><tgroup cols="5" colsep="1" rowsep="0"><colspec colnum="1" colname="col1" colwidth="31.50mm" /><colspec colnum="2" colname="col2" colwidth="31.50mm" /><colspec colnum="3" colname="col3" colwidth="31.50mm" /><colspec colnum="4" colname="col4" colwidth="31.50mm" /><colspec colnum="5" colname="col5" colwidth="31.50mm" /><thead valign="top"><row><entry namest="col1" nameend="col5" align="center">Freezing MRC-5 cells in monolayer culture in 24 well plates.</entry></row><row><entry namest="col1" nameend="col1" align="center">Support^at</entry><entry namest="col2" nameend="col4" align="center">Viability (%)^b</entry><entry namest="col5" nameend="col5" align="center">Confluence^c (days)</entry></row><row><entry namest="col1" nameend="col1" /><entry namest="col2" nameend="col2" align="center">thawing</entry><entry namest="col3" nameend="col3" align="center">24</entry><entry namest="col4" nameend="col4" align="center">48 h</entry><entry namest="col5" nameend="col5" /></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left">Free</entry><entry namest="col2" nameend="col2" align="right">85 (13)</entry><entry namest="col3" nameend="col3" align="right">42 (23)</entry><entry namest="col4" nameend="col4" align="right">25 (8)</entry><entry namest="col5" nameend="col5" align="right">-</entry></row><row><entry namest="col1" nameend="col1" align="left">Treaty</entry><entry namest="col2" nameend="col2" align="right">95 (8)</entry><entry namest="col3" nameend="col3" align="right">85 (12)</entry><entry namest="col4" nameend="col4" align="right">78 (14)</entry><entry namest="col5" nameend="col5" align="right">3-6</entry></row></tbody></tgroup><tgroup cols="5" colsep="0" rowsep="0"><colspec colnum="1" colname="col1" colwidth="31.50mm" /><colspec colnum="2" colname="col2" colwidth="31.50mm" /><colspec colnum="3" colname="col3" colwidth="31.50mm" /><colspec colnum="4" colname="col4" colwidth="31.50mm" /><colspec colnum="5" colname="col5" colwidth="31.50mm" /><tbody valign="top"><row><entry namest="col1" nameend="col5" align="justify">^at free medium or treated with collagen (100 micrograms) and fibronectin (50 micrograms)</entry></row><row><entry namest="col1" nameend="col5" align="justify">^b Viability represents the number of viable cells on the number of total cells (confluent), expressed in%</entry></row><row><entry namest="col1" nameend="col5" align="justify">^c Represents the number of days required to obtain a monolayer of confluent cells.</entry></row></tbody></tgroup></table></tables>

4.2 Results

p0050Under these conditions, the cell viability between 80 and 85%. After thawing, the cells on the treated support, occupy at least 85% of the surface available and form a complete monolayer after 24 hours of culture. The cells are spread evenly over the treated support and have an identical morphology to the control cells. Under the same conditions, but on a free medium, the cells occupy only 65 to 70% of the surface; the monolayer was formed after 48 to 72 hours. It should be recalled that the control cells (not frozen) form a monolayer within 24 hours for an initial cell density of 10⁵ MRC-5 cells / well and 2x10⁵ Vero / well.

p0051It is remarkable, that in this case, only DMSO as cryopréservant gives the best results (viability and cell spreading).

p0052Frozen cells, and then thawed, are infected after 24 hours of culture by Herpes HSV-1 and HSV-2 using the method described in Example 2.2). After 24 hours, the frozen cells show a very good sensitivity to the two virus strains, however, lower by a factor of 10X as compared to control cells. After 48 hours of incubation, the sensitivity of frozen cells is similar to that of control cells.

p0053The cells treated support can be used 24 hours after thawing but could be immediately after thawing, mixing the sample of infectious virus with the cells after the second centrifugation and culturing them. After 24 hours of incubation, the viral agent could be identified by a specific method (anti gene, DNA probe).

example 5

:

FREEZING CELLS SUSPENDED IN TUBES (CryoTubes TUBES OF CULTURE)

5.1 Methods

p0054Cells prepared according to the procedure described in Example 4.1., Are resuspended in a 7.5% DMSO solution in FCS at a cell concentration of 5x10⁶ cells / ml. It should be noted that the optimal viability is achieved in the case of MRC-5 and Vero, respectively 2x10⁶ the cell density and 5x10⁶ cells / ml cryopréservant.

p0055The cells in suspension are dispensed into tubes, either 2 ml cryotubes capacity of either 15 ml culture tubes (Falcon, Becton Dickinson) at a rate of one ml per tube. The cells are then frozen directly at -70 ° C.

p0056To thaw the cells, the tubes are heated to 37 ° to dissolve the cryopréservant. The cells are either transferred to a tube containing 14 ml of fresh serum free medium (cryovial) directly or diluted with the same volume of medium (culture tube). After centrifugation, cells are resuspended in the complete culture medium and distributed in the treated wells (collagen and fibronectin) in a proportion of 1,2x10⁵ MRC-5 cells and 2,2x10⁵ Vero cells per well.

p0057The cells are incubated at 37 ° C under 7% CO₂ in air. The medium is renewed after 24 hours.

5.2 Results

p0058Under these conditions, the cell viability is close to 84% (± 3%) for MRC-5 cells (n = 15) and 86% (± 5%) for Vero cells (n = 8). Cell viability is optimal if the density of the MRC-5 cells is at least 2x10⁶ cells / ml and the density cryopréservant Vero cells is equal to 5x10⁶ cells / ml cryopréservant.

p0059Again, the optimal spreading of the cells is obtained in the case of treated support. The density of cells used to inoculate cultures, provides a layer monocellular confluent 24 hours after thawing.

p0060This method is simple, reproducible and preserves the sensitivity of cells MRC-5 and Vero Herpes virus (HSV-1 and HSV-2). As mentioned in Example 4, it is possible to add the virus sample after thawing, during the culturing cells.