WO2004083367A2 - Lyophilised cells and a method of lyophilisation for the production of said cells - Google Patents

Abstract

The invention relates to non-living lyophilised cells from prokaryotic or eukaryotic organisms and the use thereof as standards or controls for the assay of intra-cellular nucleic acid sequences (RNA or DNA), the epigenetic modifications and the products (proteins etc.) thereof. The invention further relates to a method applied to the production of said cells.

Description

Lyophilized cells used as standards or controls for the assay of intracellular nucleic acid sequence (s), their epigenetic modifications or their products, and lyophilization process allowing the production of said cells.

The invention relates to lyophilized non-living cells derived from prokaryotic or eukaryotic organisms and their use as standards or controls for the assay of intra cellular nucleic acid sequences (RNA or DNA), their epigenetic modifications or their products (proteins). , etc). The invention also relates to a method implemented for obtaining these cells.

There is currently no reference material of complex composition allowing the comparison and therefore the analysis of the reproducibility of the assays of innovative technologies such as quantitative real-time PCR or large-scale analyzes (from a few markers to thousands / million markers in a single reaction). The availability of such reference material (standards and controls) is essential both in the field of research and in the field of biological assays in medicine or to establish reliable elements of traceability in animals and / or plants and / or transgenic plants.

In particular, there is no universal reference material:

• nor for the innovative dosages currently transferred to hospital laboratories, and possibly in the future to city laboratories for the medical world

• nor for innovative technologies which are today in the field of research but whose targeted applications will represent the dosages of tomorrow, particularly in the field of medicine.

The solution provided locally is the development of a reference sample by building up a large stock of material (cells or ribonucleic acid) stored either in nitrogen or at - 80 ° c at least, setting the the problem of keeping the cold chain and the limited quantity of equipment available.

In all cases, there is no guarantee that the dosed marker has the same dosage range between laboratories.

Furthermore, for standardization or quality control studies on the dosages of ribonucleic acids which are particularly unstable, their transport must be done in special packages while respecting the cold chain (use of carbo ice), which poses evidence of cost and legal problems, particularly in air transport.

The use of lyophilized cells overcomes these drawbacks. Indeed, lyophilized cells, containing the product to be assayed such as for example a ribonucleic acid sequence, have the following advantages:

1. They make it possible to compare and control all the stages of the assay from the extraction step of the material to be assayed (nucleic acids, their epigenetic modifications or their products). 2. They represent a clearly identified and stable material, of homogeneous composition, which, produced in sufficient quantity, provides a reference element over several years and makes it possible to judge the good quality of the assay.

3. They allow an ease of use and a limitation of transport costs: these lyophilized cells can be distributed at room temperature (around 15 ° C to 28 ° C) by simple mail regardless of the distance to be traveled in the world.

4. They make it possible to isolate ribonucleic acids, very unstable, of good quality for their assay. 5. They can serve as a reference for the comparison of results produced by laboratories and can therefore be used as standards or controls a. during the development of innovative assay techniques in the field of research allowing standardization of results such as DNA chip analyzes (Expression Profiles or Comparative Hybridization of Genomes (CGH) on chips), b. in the medical field, for biological assays of these new markers, in order to carry out quality controls

Advantageously, although in no way limiting, the lyophilized cells are obtained by implementing the method described below:

The cells to be lyophilized are chosen from; , all cells can be produced in large quantities. Advantageously, it will be a single cell line, or a mixture of cell lines from the same organism or from several eukaryotic or prokaryotic organisms. The choice of cell line or cell mixture to be lyophilized is adapted according to the marker (s) to be analyzed or assayed.

The cells are cultured in their growth medium until the exponential phase. They are then collected at this phase, then washed in a buffer type saline phosphate buffer at 2X concentration, preferably at pH 7.2, and resuspended in this buffer at the desired concentration per ampoule, for example 3.10 ⁶ cells per ml.

The ampoules are washed with acid then rinsed thoroughly, and then dried, marked and put in the oven.

The cells are then distributed in vials (1 ml / vial) and kept at + 4 ° C until the aliquoting is complete.

The ampoules are then placed in the lyophilizer, and lyophilized according to the protocol described below:

- freezing at a temperature of -50 ° C on a pre-cooled shelf,

- progressive primary drying from -50 to 0 ° C in increments

- primary vacuum between 10 and 1000 μbar, and preferably 10Oμbar - secondary drying at temperatures between 15 and 45 ° C, and preferably of the order of 25 ° C resuspension of the cells before lyophilization in a hypertonic buffer preferably chosen from the range of products below: tris, phosphate , borate, acetate, hepes, etc.

- secondary drying vacuum between 10 and 300 μbar drying of suspended cells in vaporized or air-dried form

The cell concentration can be taken between 10 ⁴ and 10 ⁹ cells per ml depending on the type of dosage.

The drying of the cells can be carried out with or without the assistance of a pressure reduction. The presence of a cryoprotector or a lyophilization protector may be necessary.

The assay can be either absolute or relative (by comparison between samples) and this whatever the methods used to express the results.

In comparative relative assays, it is advantageous to use two or more of these mixtures of lyophilized cells.

The examples below describe embodiments for assaying different products, carried out from lyophilized cells.

Example 1: Determination of the BCR ABL transcript (ribonucleic acid) by the quantitative real-time PCR technique (RQ-PCR) after reverse transcription in the lyophilized line K562.

Context: To date, there are no International Standards for RQ-PCR analyzes, particularly in the context of multi-centric protocols aimed at evaluating the efficacy of drugs. Indeed, these protocols require the comparison of the results obtained in different teams and these comparisons are made difficult by the absence of standards having a reference value. In particular, the precise dosage of the BCR ABL transcript becomes necessary to adapt the treatment according to the response to the drugs. in particular with the introduction of GLEEVEC (Novartis), a therapeutic agent that blocks the tyrosine kinase activity of the BCR ABL protein. We have developed lyophilized cells in order to have international standards.

Method: The cells of the K562 line or the Tom1 line were cultured until the exponential growth phase where they are harvested and lyophilized at a concentration of 2-3 × 10 ⁶ cells / aliquot. The bulbs used are with screw cap. These halobutyl silicone caps are washed in an industrial ethyl alcohol and air dried. They were put in a bag and autoclaves before use. The caps are fixed on the neck of the ampoules and they are freeze-dried by placing them directly on pre-cooled shelves in a Serail type freeze-dryer (CS -15 or CS-100). After lyophilization, the ampoules are rinsed with liquid nitrogen. Then the screw caps are put in place and locked by hand.

The freeze-drying cycle stopped on day 50 for the batch (01/604) is 50 days.

The vials containing the cells (nearly 1,500) were placed on shelves precooled to -50 ° C and frozen at -50 ° C for 720 minutes (12 hours). The vacuum was then applied to a level of 100 μbar and the shelves kept at -50 ° C for another 780 minutes. The temperature of the shelves was then raised to -45 ° C over a period of 25 minutes and then held at -45 ° C for 720 minutes. The temperature of the shelves is then increased to -35 ° C over 50 minutes and maintained at -35 ° C for 360 minutes. The temperature of the shelves is then increased to -25 ° C over 50 minutes and maintained at -25 ° C for 720 minutes. . The temperature of the shelves is then increased to -15 ° C over 50 minutes and maintained at -15 ° C for 360 minutes. The temperature probe indicated an inflection in the temperature of the product at -25 ° C which suggests that the primary drying was complete for the ampoule containing the probe at this time. The temperature of the shelves is then raised to 0 ° C over 75 minutes and maintained at 0 ° C for 120 minutes. The temperature of the shelves was then raised to 25 ° C over 125 minutes. The vacuum is then accentuated by SOμbar and the temperature maintained at 25 ° C for 24 hours for the second drying. The temperature of the shelves is then reduced to 22 ° C and the vacuum maintained until the bulbs are removed. Different RNA extraction techniques were tested and the quality of the extracted RNA was checked on the Bioanalyseur 2100 (Agilent). The RQ-PCR analyzes for the ABL and GUS control genes as well as for the BCR-ABL fusion transcript are carried out according to the protocol established in the EAC (Europe Against Cancer) program (European patent filed March 7, 2003 No. 03290572.1)

Once the feasibility was established in our laboratory, a strictly confidential international study was launched involving 22 expert laboratories around the world, each using their extraction, reverse transcription and amplification by quantitative PCR in real time.

Results:

1. Good quality of ribonucleic acids extracted from the extraction of lyophilized cells assessed by the Agilent Bioanalyzer 2100 (Figure 1) 2. Comparison of the expression of p210BCR-ABL and GUS transcripts in freeze-dried K562 cells versus fresh K562 cells measured by quantitative real-time PCR are quite comparable (Figure 2)

3. Preliminary study of the Stability of K562 cells freeze-dried by the accelerated degradation technique (Figure 3). The lyophilized cells maintained for 7, 21 or 30 days either at 37 ° C or at 45 ° C are stable for the assay considered for at least 30 days at 45 ° C according to the Arrhenius model. This preliminary study shows that there is no significant degradation of the gene transcripts measured by RQ PCR after 30 days at 45 ° C or less. The values obtained are comparable to those obtained on fresh cells, and demonstrate the good stability of the standards of lyophilized cells.

4. Strictly confidential international study (Figure 4). The assay is carried out on 3 dilutions (pure, 1/10, 1/100) of the material extracted from the lyophilized cells. Statistical analysis shows normal distribution allowing the definition of a reference value of the order of 90 to 100 copies of the BCR ABL transcript for 1 copy of the transcript of the ABL gene in this batch of lyophilized K562 cells.

Example 2: Analysis of the expression profile of 2 ampoules of the lyophilized K562 line.

The analysis is carried out on 10,000 nucleic acid sequences, representing as many genes, deposited on a nylon membrane. Detection uses radioactivity and the measurement is therefore absolute in this example. (Figure 5) Reproducibility is excellent, demonstrating the principle

The assay using lyophilized cells can be applied to any nucleic acid sequence or its products, extracted from cells present in a prokaryotic or eukaryotic organism, in particular plants, animals and man.

The nucleic acid sequences or their product (s) which it is desired to assay can be normal or modified in particular by mutation either naturally or artificially. These nucleic acid sequences can be present naturally in the organism, or introduced artificially by any process, in particular by transfection or transduction in cells or transgenic organisms.

The dosages permitted by the use of lyophilized cells can find application in various fields, and for example:

1. nucleic acid assay 1.1. Genomic DNA o By PCR techniques, in particular quantitative real-time PCR of one or a few nucleic acid sequences of the genome or any technique allowing precise dosing. • Analyzes by comparative genomic hybridization, in particular analyzes by "CGH array" which allow the detection and measurement of amplifications and deletions of genomic sequences between 2 samples. o Any technique resulting from the combination of the techniques mentioned above.

1.2. Assay of cellular transcripts (ribonucleic acids) o Polymerase chain reaction (PCR) analyzes after a reverse transcription step for the assay of the expression of a gene or a combination of genes and any technique allowing this assay.

• Gene expression profiles from pan-genomic or large-scale DNA chips to dedicated DNA chips with a limited number of genes. This whatever the technique used: • Whatever the support, in particular Nylon, glass or plastic with or without physical, chemical or enzymatic modifications.

• Whatever the type of nucleotide sequences deposited on this support (for example PCR products or oligo nucleotides from 10 to 100 seas);

• Whatever the detection methods, in particular radioactivity, fluorescence or colorimetry.

• Any technique resulting from the combination of the techniques mentioned above.

2. Determination of epigenetic modifications of nucleic acid sequences. Any technique, in particular high speed and / or automated, allowing the measurement of nucleic acid modifications without changing the nucleotide sequence, in particular methylation. 3. Determination of cellular proteins.

Any technique, in particular high-speed and / or automated, allowing the measurement of a cellular protein mixture, in particular the protein profiles measured by techniques involving mass spectroscopy techniques. For example MALDI-TOF mass spectrometry coupled with two-dimensional protein electrophoresis or MS / MS mass spectrometry coupled with nano-flow liquid chromatography or SELDI-TOF mass spectrometry or any other derived technique

4. Determination of post-translational modifications.

• Any technique, in particular high-speed or automated, allowing the measurement of modifications of cellular proteins after translation, in particular histones such as, for example, phosphorylation, methylation or acetylation.

The use of lyophilized cells according to the invention also makes it possible to envisage different types of measurement, and for example:

1. measurement in a pathological situation such as measurement of BCR ABL ribonucleic acid by quantitative PCR in real time (see example

1 above).

2. measurement to assess the consequences generated at the cellular level by the introduction of nucleic acid sequences or living cells o in transgenic organisms (animals or plants) o Measurement after cell or gene therapy.

Claims

1. Use of non-living lyophilized cells from prokaryotic or eukaryotic organisms for the assay of intracellular nucleic acid sequences, of their epigenetic modifications, by the technique of quantitative PCR in real time or by DNA chips. 2. Use of lyophilized cells according to claim 1 as a reference element (standard or control) 3. Use of lyophilized cells according to one of claims 1 or 2 for automated techniques of measurement in a reaction of one or more elements of cell origin this regardless of the mode of expression of the results. 4. Use of lyophilized cells according to one of claims 1 or 2 for absolute or relative assays of intracellular nucleic acid sequences, their epigenetic modifications, or their products. 5. Use of lyophilized cells according to one of claims 1 or 2 for assaying cellular markers in pathological situations 6. Use of lyophilized cells according to one of claims 1 or 2 for the assay of gene transcript by an appropriate technique. 7. A method of obtaining lyophilized cells for the implementation of claim 1 characterized in that said method comprises the following steps: a / the cells to be lyophilized are cultured in their growth medium until the exponential phase. b / said cells are collected, then washed in a buffer type saline phosphate buffer, ph 7.2, and resuspended in this buffer at the desired concentration per ampoule. c / the cells are then distributed in ampoules and kept at + 4 ° C. until the aliquotage is complete, the ampoules having been washed beforehand with acid then abundantly rinsed, dried, marked and placed in the oven. d / the ampoules are then placed in the lyophilizer, and lyophilized according to the protocol described below: - freezing at a temperature of -50 ° C on a pre-cooled shelf, - progressive primary drying from -50 to 0 ° C in increments - primary vacuum between 10 and 1000 μbar, and preferably 100 μbar - secondary drying at temperatures between 15 and 45 ° C, and preferably of the order of 25 ° C resuspension of the cells before lyophilization in a hypertonic buffer chosen from the range of products below: tris, phosphate, borate, acetate, hepes. - secondary drying vacuum between 10 and 300 μbar - drying of suspended cells in vaporized or air-dried form 8. A method of obtaining lyophilized cells for the implementation of claim 1, according to claim 7, characterized in that the cells to be lyophilized are chosen from among all cells that can be produced in large quantities 9. A method of obtaining lyophilized cells for the implementation of claim 1, according to claim 8, characterized in that said cells come from a single cell line, or from a mixture of cell lines from the same organism or several eukaryotic or prokaryotic organisms.