FR2586700A1 - Culture of a vector-containing animal cell line - Google Patents

Abstract

The present invention relates to the culture of an animal cell line using a special medium into which an animal tissue or organ is introduced in micronised form, and to the chamber whose light intensity, which is required for the multiplication and growth of the cell cultures, is characterised in being equipped with special fluorescent tubes, as well as to the possible applications of these animal cell line cultures in the pharmaceutical, veterinary, cosmetic, health food and agri-foodstuffs industries.

Description

 The present invention is characterized by the culture of an animal cell line vectorized by culture media enriched in amino acids, vitamins, oligo and macro-elements, in addition by a tissue or an organ reduced to the state of homologous micronized powder. to the tissue or organ you want to treat. Hence the originality and the novelty of the present invention.

Animal or plant cell cultures must thrive in a controlled environment.

To obtain massive cultures it is necessary to put the cells in suspension.

The part (s) assigned to these cultures must be aseptic and comprising, Vertical or horizontal laminar flow hood with total or partial aspiration or expulsion. It is therefore useful and necessary to make a serious choice with regard to the various products to be handled to be protected. Microorganisms and pathogens if these are used should also be taken into account when choosing this type of material. The laminar flows benefit from a classification by category C, this one with regard to the manipulations and cultures that I have to carry out, then also with regard to the micro-organisms that I could be called upon to use eventually. The media used for cell cultures require strict quality control, hence the need for a stable formulation.

The media and sera will therefore have to be of high quality.

The cells used come from the "American type collection (ATCC)".

The laboratory technique used is simple but requires a lot of care and attention. Cell culture is thus defined by 1) The cell line.

2) The culture medium.

3) The culture technique (monolayer suspension), (I chose suspension culture).

4) The duration of production, in popularization production.

 There are three types of cells for cell cultures, primary cells, cell lines for cells of limited duration and long duration, therefore with a large number of passages.

Primary cells are cells taken directly from specific organs or tissues. They are considered as such until the first secondary culture, then they become a cell line. However, I use these primary cells as an enriching factor under certain very stringent conditions explained in the culture technique, which represents an innovation.

The dull cell line is thus defined by cells capable of supporting an indefinite number of in-vitro passages, a cell must generally be able to withstand at least 70 passages from the primary strain before it can be designated as a "cell line. "a line of limited duration can undergo only a limited number of passages before dying. It must be pointed out that a primary cell before becoming a cell line must accept certain modifications of its strain, which can be carried out slowly or very quickly after a few passages. In cell lines, the most well-known modification or, more precisely, the difference is that of the presence of an abnormal number of chromosomes and this passage whereas at the start all the primary cell lines comprise a normal number of chromosomes remains indicated or described for the name of "transformation".

An important peculiarity is that primary cells cannot be reused or multiplied, they can be considered for single use (reagent), on the other hand, cell lines have a power of multiplication. I work the cell line safely when I have made 10 passages from the primary cell and these cultures in suspension because they are easier to handle but more difficult to observe microscopically. This suspension can only be obtained using a support. I use in this case the "superbead" from "Flow Laboratories". On the other hand, certain cells do not need this support. It should be noted that some cells may have behaviors that are not similar with different culture modes, this is why and as I mentioned above, it is preferable to work with cells in suspension than single-layer with regard to the results which may vary from one culture technique to another.

Admittedly, the monolayers are more easily observable, especially when there may be cytological modifications, more than they are more easily achieved by fixing in an independent manner.

 It is also interesting to note that certain cell lines tend to proliferate in excess, while others have no particular characteristic until the stage where they become confluent.

A very important note never to work in the production laboratory with two cell lines at the same time, in order to avoid the risk of cross contamination, all the premises must also be partitioned. These lines in culture must be continuously monitored day and night with regular checks, because they split every 3, 4 or 5 days, so there is no rest day possible, we can of course choose lines cells which can be split irregularly which reduces the above taxation but can cause risks and inconveniences. When the cell line is in culture, it is compulsory to have a material for 7 consecutive days, this is very important. The culture media are generally defined by the cell line used. In our case, these were chosen according to this criterion, explanations provided in the manufacturing technique.

The water used for cell cultures is purified water by reverse osmosis and ultra pure water. The bases, serums, additives, buffers are selected, defined with criteria of purity and depending on the cell line chosen. -All sterilization is carried out by membrane filtration, pyrogen-free filter for water and media as well. The sera used are also selected with regard to the cell line with compatibility properties, the additives incorporated into the media according to an aseptic technique, the same buffers. Since the storage temperature of these products complies with the specifications, the laminar flow enclosure must include a passage for the gas through an appropriate sterilizing filter. The C o 2 incubator if necessary, however very useful, when the 'sodium bicarbonate is used as a buffer. All plate shaker equipment, filtration and pipetting system, culture plates and dishes, glassware, must be the best, attention to maintain aseptic, all bottles must be rinsed with ultra pure water , previously cleaned with a detergent "Flow 7x" specially studied for cell cultures.

The bottles can be for single use, I use this technique.

In summary, a large stock must be put in place, since a culture started cannot stop.

The cell strains can be frozen in liquid nitrogen, this means avoids the risk of loss by infection or abnormal genetic modification, it is also a means of control, so one can after 10 frozen passages a sample of the culture giving the possibility of controlling a modification always possible for the continuation of the culture then of re-examining then the initial sample taken, Thus by proceeding in stages it is possible to detect where can this modification be situated.

I encountered in my experiments the problem of slow growth which can and must be avoided, it is then necessary necessarily to check the quality of the water, the buffering capacity, to check the medium, the PH, the osmosality, the serum, growth medium, presence of endotoxins, contamination, concentration, method of washing glass bottles, toxicity of plastic bottles, filters of laminar flow equipment, temperature of incubator, number of passages, growth rate and other needs. The most formidable enemy is contamination and any contaminated culture must be discarded. If despite these controls the contamination repeats, it is necessary to carefully check the incubators, the autoclave, the laminar flow chambers, the filtration processes and even the storage containers: a contaminated cell line, as it is indicated above must be discarded, but have no replacement strain available, we can try to kill a decontamination by washing the line, then re-implant it in an antibiotic solution. This cell line thus washed and reimplanted, will be rinsed and transplanted in a medium this time without antibiotic. This process which I applied several times as experimental tests was repeated for 8 passages until obtaining without contamination did not not always entirely satisfactory, because certain decontaminations do not always succeed, say 1 for 4 which gives a negative result, but the test can be attempted. It is therefore absolutely necessary to follow all the recommendations indicated in this description to carry out a cell culture.

Control technique for mycoplasma contamination.

Different samples are taken from the culture after each passage for the detection of mycoplasmas by staining with "fluorochrome!". The cells are incubated in dishes treated for cell culture for 5 days. To stain with fluorochrome, it is necessary to fix the cells .

The colored preparation is then examined under a microscope to detect any extra-nuclear fluorescence.

Hemocytometer control technique.

Do not overfill the chamber, allow the cell suspension to settle by capillarity. Count the cells in the central square and in the four corner squares. Cells touching the upper right lines of the perimeter will not be counted, those touching the lower left lines will be counted. Dead cells stain blue, the sample of the diluted cell suspension being previously stained with "Blue TRYPAN". The number of cells in a large square will be: N / 5, the number of cells per ml of diluted suspension: 104 N, and the number of cells / Nl of undiluted suspension 104 y. 5
Living cell counseling technique.

Cool the suspension in an ice-water bath and add glycerin or dimethylsulphoxide (D M S O) to it.

I use D M S O to obtain a final concentration of 10%. The concentration of cells in suspension should reach 2.5 - 4.0 x 106 viable cells / ml.

Transfer the suspension from an ice water bath using a syringe with an 18 g needle (pink) to a sterile ampoule, then seal the ampoule, then put the ampoule in a box. 5 x 10 cm thick polystyrene. Close the box and put it in the freezer at -70 ° C for 2 hours. The cooling rate is 1'C per minute. Then keep the ampoule alone in a container with liquid nitrogen.

A technique for restoring frozen cells.

When removing the bulb from the container, wear gloves and a mask because it is to be feared that a badly sealed or unsealed bulb may explode due to thermal shock.

Thaw the contents of the ampoule in a 37 C water bath, clean the outside of this ampoule well with 70% alcohol and allow to dry at room temperature.

Break the end of the vial and transfer the contents of the vial sterile using a syringe fitted with an 18 gr needle (pink cone) into a culture flask if this content is intended for the latter. .

If the content is intended for a culture, add in the culture medium sufficient to dilute the DMS 0 to 1 / 10th in the sample reception flask. If the culture requires a weaker suspension, centrifuge the suspension at speed of 200 g / 6 minutes before being resuspended in a new culture medium. Very often I noticed that it was necessary in the latter case to incubate at 37 C, that an atmosphere of 5% of C 02, 95% of air is useful for obtaining an adequate Ph for the growth. After 24 hours it is necessary to replace the medium with a new one and put the cells back into incubation.

Culture in suspension.

The cultures in suspension are sent by the supplier of the strains at the rate of 1 × 10 6 viable cells per ml. Upon receipt, they must be incubated at 37 C. I checked the viability of the cells by staining, but it must first be brought to room temperature. All cell cultures shipped by suppliers must include the number of passages -Date of inoculation -Date of culture - growth medium confluence and count. The shipping flasks are completely filled with culture medium, they can be reused after decanting for, for example, subcultures.

Method of sampling an animal tissue or organ for vectorization in cell culture.

1) Anesthetize the animal with intramuscular or subperitoneal injection with membutal (1 ml per 2 kgs) 2) Disinfect the skin with alcohol, incise with sterile instruments to access the desired organ or tissue.

3) Remove tissue or organ with new set of sterile instruments. Place these in a sterile bottle or a sterile petri dish (large diameter) as well. (The tissues or organs are cut into pieces 2 to 3 mm square.

4) Transfer these pieces into an Erlenmeyer flask containing a magnetic bar
5) Introduce into the Erlenmeyer flask an Earle solution (without calcium and magné
sium) which is a balanced saline solution and a streptomycin solution
(4% penicillin, percentage by volume).

6) Stir and wash 2 times with the same solutions. Throw after
wash these solutions, except sampling.

7) Then add 10 ml per gram of Earle medium without calcium or magnesium,
to which must be added 20 ml of trypsin and 4% streptomicin, penicillin
percentage by volume. Shake everything for about 15 minutes, remove the li
what supernatant. Let rest.

8) Take the sample aseptically from the bottle and wash it twice with
ultra pure water in which 2% newborn calf serum is added
(percentage by volume).

9) Take the sample aseptically from the bottle, transfer it to one or
several sterile petri dishes, dry in an oven at 40
10) After drying, grind and reduce to micronized powder to 0.2 micron with
apparatus under sterile control, store in the freezer at - 20'C.

11) It is possible to freeze-dry the product without seeking a reduction
micronized after grinding.

12) The sample is ready to be introduced into cell cultures,
Manufacturing technique for vector cell lines.

1) Collect the organ or tissue from the animal according to the conditions indicated
by the technique or method of sampling an animal tissue or organ
for vectorization in cell culture to weigh after collection
the organ or tissue. The manufacturing technique therefore resumes from the n of
reference 6 of the sampling method.

2) Add to the bottle containing the sample 10 ml per gram of medium
Earle always free of calcium and magnesium, then 10 Z (percentage by volume)
of trypsin, phenol red which must be at 2.50 / (W / V) 1250, also
4 S (W / V) of streptomicin / penicillin.

Shake the bottle for a few minutes at reduced speed, let stand, remove
the supernatant.

3) Add 20 ml of trypsin per gram to the sample container
and shake for 15 min at 37 ° C. Let file and reject the
supernatant, which removes red blood cells.

4) Add trypsin in solution (20 ml per Gr), stir at 37 C pendar, t
30 minutes approximately. Let the large debris from the sample stand and decant the
cell suspension through sterile gauze in a centrifuge tube,
add approximately 10% (W / v) of newborn calf serum to stop the action) trypsin.

5) It is necessary to reject this trypsination, with the large debris remaining in the bottle in order to completely eliminate the cells and obtain only cellular fibers.

6) Centrifuge the suspension at approximately 200 g for 10 minutes and discard the supernatant.

7) Resuspend the cells in culture medium "Eagle's minimum essential medium for suspension cultures.

8) Count the viable cells with a colored indicator (Tripan
Blue) and hemocytometer.

9) Dilute the suspension in a BME Hanks salt medium at the rate of 2 to 3 × 10 6 cells per ml. Add to this medium 10% fetal calf serum (W / V) and a penicillin solution (streptomycin, 2% at 5,000 / ml for penicillin and 5,000 microgram / ml for streptomycin, then 10 ml of a 20% solution of the micronized sample.

 10) Incubate for 96 hours at 37 ° C.

 11) After incubation replace the BME medium with Hanks salts with another BME medium but with Earle salts to which 10% fetal calf serum is added, plus a 2% solution of antibiotic, penicillin / streptomycin, and 20 ml of a 30% solution of the micronized sample.

12) Incubate again in the incubator for 72 hours at 37 ° C.

 13) The C 02 incubator must be set at 37 ° C and 95% air and 5% C o2.

 14) After incubation for 72 hours at 37 ° C., divide the suspension into X equal parts, introduce (these parts by volume and weight of the container with its contents) into Erlenmeyer flasks, add 200 ml of BME medium with Parle salts, 10 % fetal calf serum, 5% HAM nutrient medium, maintain these cultures in a sterile room, with a percentage of C 2 equal to and not greater than 5%. Light by fluorescent tubes of the big lux mazda type must illuminate the culture room in an identical way to daylight from the day of sunrise until sunset. Adjust everything by clock. The room temperature must be 37 C, the air conditioning and ventilated (air conditioning) must be sterile. These conditions will allow the final product to succeed in what is necessary and useful for growth, the multiplication of cells of the cell line.

15) This last cell culture represents a passage, it is necessary to obtain a final product to carry out 10 passages.

16) At each passage, double the media, for the solutions, nutrient medium and powder sample explanations given below as an example for the
Earle salts progression will be 200-400-800-1,600-3 200-6,400 etc.

 For fetal calf serum increase by 5, that is: 10-15-20-25-30-35-40 45-50.

For the nutrient medium increase by 5, ie 5-10-15-20-25-30-35-40-45-50.

For micronized powder sampling, this will only be added from the 4th pass, 6th pass, 8th pass.

On the 4th pass 20 ml of a 40 Z solution
On the 6th pass 40 ml of a 40% solution
On the 8th passage 80 ml of a 40% solution 17) The time required between each passage is 4 days.

18) Every 4 days, the contents of the containers must necessarily be transferred to other suitable containers already containing the solutions and lined ready media. It is not necessary for handling reasons to take containers of large volume it is enough to carry out each time the useful calculations. The final product for a production of 110 liters must be distributed in 110 vials of 1 liter.

19) During the period of time between each pass, ie 4 days, the cultures must be shaken every day for 2 hours for 5 minutes, day and night.

20) At the end of cell production, aseptically centrifuge each vial in a centrifuge tube, leaving approximately 10 ml of the fluvial medium with the cells in suspension which are placed in cell mass in these tubes.

Add (after taking everything and placing in a sterile 50 ml wide-opening bottle) 30 ml of ultra pure water, stir for approximately 2 minutes.

At the end of the agitation, subject the cells to the "ultrasound" technique until the cytoplasmic membrane bursts.

21) Collect all of the solution which has been passed through the ultrasound technique and transfer it sterile to a large container to be stored in a freezer at -20 C.

 2?) This product can be frozen up to 160 C if you want true freezing and storage.

23) All the culture conditions have been met to obtain a biologically pure end product.

24) All control operations are carried out as specified in this request, every day, with sampling taken at each pass.

25) The room for the culture of a cell line was created in Rennes with a particular arrangement, which required by the Mazda Company a particular study of fluorescent tubes. The lighting, the intensity, the colors, were studied by myself for the growth and the multiplication of the cells, according to the brightness of the day and the night, the whole regulated by clock system with regard to the seasons, sun, moon, 160 tubes were required, air conditioning, air, Co2 were the subject of an identical study.

To maintain the necessary balance in the room, ie 95% air and 5% Co2 with a temperature of 37 ° C, the air coming from the outside was filtered, then air-conditioned, and taken up by laminar flow blowing forward.

Another laminar flow was in the room for handling media, solutions, and cell cultures.

 Growth media for cell cultures and other products used for cultures, organ and tissue samples.

1) Minimum essential medium for suspension cultures, add 10% fetal calf serum.

2) Eagle base medium with Earle salts containing 10% fetal calf (serum) 2 m M glutamine, 1% non-essential amino acids.

3) Minimum essential medium with Hanks salts containing 10% fetal calf (serum), 2 mM glutamine, 1% non-essential amino acids.

4) Medium (nutritive mixture of HAM) association of amino acids, trace elements and macro elements, without sodium bicarbonate and L glutamine.

5) Eagle's minimum essential medium for suspension crops.

6) Newborn calf serum free from mycoplasmas and viruses, to be used at a dose of 2 Z (W / V) to be stored in the freezer at -20 C (quality certificate required).

 Conditions required for these culture media.

Must be sterilized by filtration on 0.2 micron membrane.

For powdered products be taken up in ultra-pure water and sterilized by filtration.

For L. glutamine, add it in the middle only at the time of use. Do not store at temperatures above -10 ° C, due to the instability of this product at higher temperatures.

The water must be pyrogen-free and contain less than 1 PPM of solid particles and less than 0.1 PPM of heavy metals. Water purification must be carried out by reverse osmosis or is deionized, then sterilized by filtration through a 0.2 micron membrane.

TRYPSINE must be magnesium-free, calcium-free and phenol red, frozen shipment with ice-cream.

Blue trypan, 0.5% solution (W / V).

Sodium bicarbonate, 7.5% solution (W / V) (quote here, but little used, preferable to act with HEPES buffer).

Amino essential acids, in solution.

HEPES buffer, 1 M (Flow specialty) PH 7.2 - 7.4 at 37 C, it can be added sodium bicarbonate to this buffer, but the concentration should not exceed 0.85 gr / L.

Antibiotics: In solution to avoid toxicity with antibiotics for cell cultures in suspension. Do not exceed for penicillin and streptomycin (the two antibiotics chosen) and more particularly when the media contain serum.

Penicillin in units per ml 100 u / ml, streptomycin in micrograms per ml 100 / ml. An increase in these units can cause a reduction in the multiplication of cells.

Serum
When stored at -20 ° C in a freezer, the serum must be free from bacteriophages and virus mycoplasmas. This very important guarantee must be the subject of a certificate given the necessity of the serum for cell growth and survival.

NOTE: There are prepared media, containing all of these products except trypsin. I used both to know prepared or not.

 Table concerning the storage of the products which have just been listed when these are purchased separately.

Antibiotics stored at - 20'C duration 1 year
L glutamine stored at - 20'C duration 1 year
Concentrated vitamins stored at - 20'C duration 1 year
Essential amino acids stored at - 20 C duration 1 year serum stored at - 20'C duration 1 year
Use of detergent for laboratory glassware and other instruments necessary for cell culture.

Conditions: Store at room temperature
PH close to normal 7
Biodegradable
Dry in minutes
Maximum solubility
Dispersion of suspended solids and deflocculation.

Safe for glass
No toxicity
Very effective
Chemically safe to handle
No opposition to the development of cell cultures in tubes, flasks, boxes.

To be used at a dose of 5 to 10% for conventional washing of glassware.

NOTE: If there is oily or oily dirt, leave in contact overnight then heat the solution to the limit of boiling then discard, then pass into ultra pure water in an ultrasonic tank for 30 minutes.

Previously rinse thoroughly in five rinses then 3 rinses in ultra pure water.

Notes on glassware
Wash with 5% detergent solution.

All glassware must be autoclaved at 130 C for 10 minutes, but must first be soaked in an ultrasonic tank with ultra pure water for 30 minutes.

Means of controlling batches of cell cultures at the start of the culture and in finished production before bursting of the membrane with large equipment used
Take 100 ml from the final production, distribute into 4 sterile bottles of 25 ml each.

Incubate for 7 days at 31 ° C.

Take 10 ml from each bottle.

Transplant into a soy tryptone medium (broth) and a thioglicolic acid broth (these broths can be bought prepared) then put back into incubation for 10 days at 31 ° C.

Which gives 8 media each receiving 10 ml or 8 vials.

Each bottle is examined daily for biological contamination.

 It should be noted that during the multiplication of the cells until the final production, checks are carried out to detect a possible contamination by the mycoplasmas or the appearance of a ladle, it is necessary for an additional check to carry out this- here on the end of production.

If a doubtful result is obtained for this purpose, it is necessary to repeat the same tests on new bottles. The slightest contamination detected, the whole lot must be destroyed.

Control the PH throughout the multiplication and this until the final product, this every 2 days and 2 times a day.

Determine each day and until the final product the size of the cells, their morphology, the presence of anomaly obliges the immediate cessation of production and research thereof.

Control of the number of cells per ml by hemocytometer every three days.

Control of the appearance of cell culture.

Amino acid determination by special kit.

Detection of mycoplasmas by colorimeter.

Control system in the event of cellular modification by prevention in freezing liquid nitrogen after 10 passages, which gives the possibility of having a reference sample.

Control of the room (s) where the cell cultures are carried out by placing boxes of gelatinous petri dishes (these boxes can be purchased ready to use) at the corners. Any contamination must stop the cultures.

The laminar flow must be class III (vertical) for the events necessary for cultivation.

The air must be ventilated by another laminar flow, air forced towards the front.

Air conditioning by air conditioner controlled by clock.

Very important originality
The lighting controlled by clock respecting precisely the lighting of the day and the night by increasing and decreasing action according to the sunrise and the sunset (the brightness) and this by special tubes type mazda adapted to the spectrum of the light in color.

 Co2 incubator adjustable in temperature, in Co2 opposite the external one, humidity.

 Oven at 37 C bacteriological type.

 Drying oven up to 180 ', ventilated.

 Freezing equipment for mini-color type oxygenated products (freezing at -70 * C).

 Vacuum pump and small laboratory compressor.

 Microscope with television screen (monitoring) and (microphotographic equipment.

Autoclave on foot (current model)
Germination oven.

 Ultrasound material for bursting cell membranes.

Applications of vectorized animal cell cultures
Vectorized animal cell cultures find their applications in the following industries
Pharmaceuticals, veterinary products, cosmetics, food, dietetics.

In fact, these cultures cultivated on special media into which a tissue or an organ reduced to the micronized state is introduced, coming from a sample from a living anesthetized animal, sample chosen opposite, therefore homologous with a tissue or a organ to be treated, gives the opening 9 of numerous possibilities for preventive or curative actions which cannot be located here because they can only be defined positively or negatively in relation to demand. The cultured cell line is itself obtained, according to the technique of animal cell culture, from the same sample, however, under specific culture conditions. This sampling technique with its line representing a new character, to which is added the vector effect.

Important note
Vector cell plant cultures.

 The sample, instead of being an animal sample, is a homologous plant tissue or organ.

 The culture media are not identical but composed of micro and macro-elements therefore of nutritive solution.

 The manufacturing technique is the same as for vectorized animal cell culture.

Claims (5)

 1) Animal cell line cultivated with a special medium into which an animal tissue or organ is introduced in micronized form.  2) The cultured cell line, characterized by the tissue or organ from the same animal by sampling, hence the vector effect.  3) The vector cell cultures are characterized by the culture bringing together all the aseptic conditions necessary to carry out these cultures without contamination, namely temperature at 37 ° C., useful contribution for development of cells in light intensity, supply of sterile air and renew at 95% and 5% Co2, all regulated by a clock system.  4) The room for the light intensity, necessary for the multiplication and growth of cell cultures, is characterized by the equipment of 160 special fluorescent tubes studied by MAZDA, illuminating cell cultures at all levels on the one hand, d on the other hand this light intensity of day and night, that is to say respecting the hours of sunrise, its setting for the day, the moon ditto or the degree of darkness of the night. This action controlled by a clock system is therefore increasing, decreasing, the cycle of sunrise and sunset is thus respected.  5) The possible applications characterized by these vector cultures are very vast and can only be established in relation to demand in the pharmaceutical, veterinary and cosmetic industries. dietetics, food.