GB2408750A

GB2408750A - Medium and method for preserving biological material

Info

Publication number: GB2408750A
Application number: GB0327933A
Authority: GB
Inventors: Arthur A Codd
Original assignee: Individual
Current assignee: Individual
Priority date: 2003-12-02
Filing date: 2003-12-02
Publication date: 2005-06-08
Anticipated expiration: 2023-12-02
Also published as: GB0327933D0; GB2408750B

Abstract

A medium for preserving biological material such as bacteria or yeasts comprising water, 5 to 30 wt% of one or more polyhydric or sugar alcohols and 5-30 wt% one or more colloidal or soluble crystalloid materials and buffered to pH 6.5-7.5. The sugar alcohols may be adonitol or sorbitol. The colloidal or soluble crystalloid materials may be bovine serum albumin (BSA), ovalbumin, carboxymethyl cellulose, gum arabic, alginate, gelatin, DEAE, Ficoll<RTM>, polyvinylpyrrolidine. The medium may also contain up to 15 wt% soluble peptide digest or hydrolysed or partially hydrolysed gelatin. Also claimed is a method for preserving biological material by dispersing the material in said medium and drying the resulting dispersion above the triple point of the mixture. Drops of the dispersion may be dispensed onto a hydrophobic material and the drying may be carried out in sealed containers.

Description

Preservation of Biological Material This invention relates to compositions

and methods for use in the preservation of biological material, particularly for the long term storage of viruses, bacteriophages, prokaryotes, eukaryotes and their sub-cellular components, including enzymes, antigens, membranes, nuclei, plasmids, DNA and RNA.

Many means have been used for the preservation of biological materials, particularly living organisms, beginning with the drying of broth cultures of micro-organisms on silk threads. Freeze-drying remains the principle means of preservation of micro-organisms for distribution of strains from national culture collections; however, this procedure is generally associated with large initial losses in viability leading to difficulties in producing materials containing defined numbers of live cells. Other common methods include Cryo-preservation on beads, or in vials, at -80 C in freezers, or at -192-C in liquid nitrogen; however such methods have many drawbacks, including the need for specialist storage equipment to maintain the necessary low temperatures. Materials stored under such conditions are also difficult to transport and the freezing process often results in significant physical damage to the stored material. Other methods based on drying include spray drying organisms dispersed in milk to form a powder. This powder is then mixed with further dried milk powder and enclosed in gelatine capsules.

EP-A-1003837 discloses a composition for preserving micro- organisms in specific amounts, comprising at least a substance capable of forming the skeleton of lens shaped lozenges, at least a saturating substance and predetermined and reproduceable amounts of micro-organisms. The compositions are formed into pellets by drying under vacuum at a temperature of less than 10-C.

WO-A-01/05941 discloses a composition for preserving viable microorganisms, cells or tissues comprising (a) a preservative combination of (I) a non-reducing disaccharide and (ii) a bulking agent; and (b) a buffer. Once the biological material has been combined with the composition, it is preserved by drying at above the triple point of water.

The present invention provides a preserving medium for use in preserving biological material, comprising water, 5 to 30 wt% of one or more polyhydric or sugar alcohols and 5 to 30 wt% of one or more colloidal or soluble crystalloid materials, and being buffered to a pH of 6.5 to 7.5.

The present invention also provides a method for preserving biological material comprising: (1) dispersing the biological material in a preserving medium of the present invention, and (2) drying the resulting dispersion above the triple point of the mixture, at a temperature of -5 C to C. The preserving medium of the present invention may comprise one or more additional liquid components selected from DMSO, ethylene glycol or glycerol, up to a total of 30 v%; however, it is preferred that water is the only liquid ingredient.

The one or more polyhydric or sugar alcohols for use in the preserving medium of the present invention is preferably selected from adonitol, sorbitol and mixtures thereof, sorbitol being the most preferred. A preferred concentration range for the one or more polyhydric or sugar alcohols in the preserving medium of the present invention is from 5 to 20 wt%, more preferably from 7 to 15 we%, for example 8.3 wt%.

The one or more colloidal or soluble crystalloid materials for use in the preserving medium of the present invention is preferably selected from bovine serum albumin, ovalbumin, carboxymethyl cellulose, gum arable, alginate, gelatin, DEAE, =0 Ficol, polyvinylpyrrolidone and mixtures thereof. The most preferred colloidal or soluble crystalloid material for use in the preserving medium of the present invention is polyvinylpyrrolidone having an average molecular weight of 2000 to 15000; more preferably an average molecular weight of 30000 to 80000, and most preferably an average molecular weight of 40000.

A preferred concentration range of the one or more colloidal or soluble crystalloid material in the preserving medium of the present invention is from 5 to 20 wt%, more preferably from 7 to 15 wt%, for example 8.3 wt%.

The preserving medium of the present invention preferably further comprises up to 15 wt% of a soluble peptide digest. Any conventional peptide digest (for example one prepared from meat or soya) may be used; however, it is particularly preferred to use a commercially available peptone. A preferred concentration range of the soluble peptide digest material in the preserving medium of the present invention is from 5 to 10 wt%, for example 8.3 wt%.

The preserving medium of the present invention preferably further comprises up to 12 wt% hydrolyzed or partially hydrolysed gelatin. Any source of gelatin may be used, as may any conventional hydrolytic procedure. A preferred partially hydrolyzed gelatin for use in the preserving medium of the present invention has a low viscosity at room temperature (20-22 C) and a high viscosity (but remains liquid) at 4 C in a wt% aqueous solution. A preferred weight range of hydrolysed or partially hydrolyzed gelatin for use in the preserving medium of the present invention is from 5 to 10 wt%, for example 8.3 wt%.

Any conventional buffering agent may be used in the preserving medium of the present invention. Preferred buffering agents include sodium hydrogen carbonate and dipotassium hydrogen phosphate. Dipotassium hydrogen phosphate is a particularly preferred buffering agent for use in the preserving medium of the present invention. The preferred pH range of the preserving medium of the present invention is from 7 to 7.5. If necessary, sodium hydroxide may be used to adjust the pH of the preserving medium to the preferred range.

The preserving medium of the present invention may optionally comprise up to 0.5 wt% of one or more additional components selected from coloring agents, reductants, anti-oxidants, detoxicants, viscosity modifying components, growth factors, substrates, markers and salts.

The preserving medium of the present invention preferably has a viscosity of from 1 - 10 mPa.s at 25 C and a viscosity of from 500 to 1500 (more preferably 900 to 1100) mPa.s at 4 C. Before use the preserving medium of the present invention is preferably sterilized. Preferred means of sterilization include autoclaving, tyndallization and filtration.

Before use, the preserving medium of the present invention is preferably stored in aliquots. The preferred storage temperature is -18 C and the preserving medium is preferably not subjected to repeated freeze/thaw cycles.

In the method of the present invention, the biological material to be preserved is dispersed in the preserving medium of the present invention at a preferred concentration of up to wt%, more preferably up to 10 wt%. When the biological material to be preserved is live bacterial cells, the preferred concentration range is from 250 colony forming units (CFU) per ml of preserving medium to 2.5 x 10l CFU per ml.

In the method of the present invention, following dispersal of the biological material to be preserved, the preserving medium of the invention containing biological material may be dispensed into containers (such as vials) for drying.

Alternatively, the preserving medium containing biological material may be spread as a film on a membrane or wetable surface for drying. In a preferred alternative, drops of the preserving medium containing biological material may be dispensed onto a hydrophobic surface for drying. -s-

Dispensing of the preserving medium of the invention containing dispersed biological material is preferably carried out at room temperature.

Preferred hydrophobic surfaces for use in the method of the present invention include parafilm, waxed paper; surfaces coated with paraffin wax, oil or Vaseline; silconised glass and plastics such as polythene, polystyrene or teflon.

In the method of the present invention, preserving medium containing biological material may be dispensed on to a hydrophobic surface by spraying small droplets to produce small spherical particles after drying, or by dispensing predetermined volumes on to the hydrophobic surface. Where predetermined volumes are dispensed onto the hydrophobic surface, the preferred volume is from 1 pi to 50 pi, more preferably 40 pi.

Dispersal of drops of the preserving medium in this manner should result in the production of discs of dried material following drying.

In the drying step of the method of the present invention, it is preferred that the preserving medium containing biological material should neither boil nor freeze. Drying may be carried out under reduced pressure or at atmospheric pressure.

A preferred range of drying temperatures for use in the method of the present invention is from 3 to 5 C, more preferably 4 C. In the method of the present invention, drying is preferably carried out in sealed containers and means are preferably provided for removing water from the atmosphere surrounding the materials to be dried. Suitable means for removing water from the atmosphere during drying include the provision of a stream of dry gas and the use of a freezing trap.

A preferred means for removing water from the atmosphere during drying is the use of desiccant materials, for example silica gel or phosphorous pentoxide.

In the method of the present invention, where drying is carried out in the presence of a desiccating agent, the desiccating agent is preferably changed after 24 hours, and may optionally be changed again as required during the drying process.

In the method of the present invention, where drying is carried out in a sealed container, the atmosphere therein may be air or the air may be partially or wholly replaced by one or more inert gases, for example nitrogen or argon.

In the method of the present invention, drying is preferably carried out until the moisture content is reduced to 6 wt% or less, preferably from 4 to 6 %. Drying is preferably completed within 7 days.

Following drying, the dried materials produced by the process of the present invention are preferably stored under dry conditions, most preferably in the presence of desiccating agents, for example silica gel.

The dried materials produced by the method of the present invention may be held at room temperature (or even higher temperatures) for periods of up to approximately 10 days; however, for long-term storage, the dried materials are preferable stored at temperature of 4 C or lower, most preferably at -18-C.

Following storage the dried materials prepared by the method of the present invention may be reconstituted by any conventional means, including dispersal in liquid medium or reconstitution on solid agar plates.

Dried materials produced by the method of the present invention, particularly when prepared in the form of discs, may be of particular use in the following applications; the storage of collections of type strains as an alternative to freeze-drying; the collection and storage of strains for research; the preparation of seed lots of frequently used cultures to avoid the necessity of frequent subculture; the preparation of marker organisms, with or without competitor organisms, at a defined count for the control of laboratory standard operating procedures where the results are expressed numerically or at a presence-absence level; the preparation of selected strains at a level suitable for antibiotic assay controls; the preparation of selected strains at a suitable level for testing biocides; the preparation of panels of organisms, either singly or as mixtures, for the testing of media, evaluation of methods, training, individual performance assement, internal quality assurance and external quality assurances schemes; the simulation of clinical, food, water and environmental specimens by incorporation or spiking of appropriate carrier materials with defined inocula.

the preservation of seed lots or starter cultures of organisms for industrial purposes, such as for fermentation or vaccine production; the preservation of non-viable organisms as particles for microscopy, immunodetection, antigen and controls for PCR; the quantitive preservation of specialized proteins in a dried form, such as enzymes, antibiotics and lectins; the preservation of biologically active materials attached to a solid phase.

The preserving medium and process of the present invention are particularly useful for the preservation of bacteria and yeasts for example Staphylococcus aureus, Staphylococcus epidermidis, Bacillus cereus, Bacillus subtilis, Wisteria monocytogenes, Enterococcus faecalis, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus pneumonias, Enterococcus faecium, Clostridium perfringens, Clostridium sporogenes, Escherichia coil, (including 0157), Salmonella sp, Pseudomonas aeruginosa, Klebsiella so, Citrobacter freundii, Enterobacter cloacae, Proteus so, Campylobacter so, Vibrio parahaemolyticus, Acinetobacter so, Serratia marcescens, Legionella so., Haemophilus influenzas, Bordetella pertussis, Burkholderia cepacia, Neisseria menigitidis, Neisseria gonorrhocae, Bacteroides sp. Sacchromyces sp Candida albicans and Aspergillus Niger.

Examples

Example 1

A preserving medium was prepared according to the following formula: Polyvinylpyrrolidone 40000mw (SIGMA) 83g Peptone (Oxoid) 83g Sorbitol (SIGMA) 83g Partially Hydrolysed Gelatin (prepared as set out below) 85g (in 250 ml water) HPO4 (SIGMA) lg Deionised Water to 1 ltr All of the above materials were mixed at room temperature and the resulting preserving medium was sterilized by free steaming for 10 minutes on 3 successive days (Tyndallization).

The medium was dispensed into 16 ml aliquots and stored at -18 C (ie frozen in a domestic grade freezer).

Partially hydrolysed gelatin was prepared as follows: 85g of granulated gelatin (SIGMA) was dissolved in 250ml deionised water.

The pH of the solution was adjusted to 3 by dropwise addition of N hydrochloric acid.

Sterilsation was carried out by free steaming for 10 minutes.

lg of protease type XVIII (from Rhizopus species, sigma P-5027) was dissolved in 10ml of deionized water and sterilised by filtration.

The dissolved protease was added to the gelatin solution and mixed throughly, followed by incubation at 37 C for 20 hours.

The mixture was cooled to room temperature, and then to 4 - C, and the viscosity under each condition was noted. If the mixture was viscous at room temperature and/or set at 4 - C, further incubation at 37 C was carried out and the above cooling was repeated until a mixture was obtained that had water-like viscosity at room temperature and was viscous (but did not set) at 4-C.

The protease enzyme was inactivated by a free steaming for minutes and any precipitate formed by coagulation of the enzyme was removed by sedimentation.

Example 2

(1) E. cold NCTC 9001 was inoculated onto Colombia base blood agar and incubated at 37 C for approximately 20 hours.

(2) 16.4 mg of E. cold culture was removed and added to 200 pl Brain Heart Infusion Broth (BHI) and a further 1440 pl of BHI was added and mixed thoroughly (3) 100 pi of the suspension was transferred into 10 ml of BHI to produce the equivalent of 1 mg E. cold cells (approximately 4 x 108 CFU) in 10000 pi.

(4) Aliquots of 600, 120 and 24 ul respectively of the above suspension were added to three 20 ml aliquots of the preserving medium of Example 1 (warmed to room temperature). The 3 suspensions were vigorously mixed.

(5) 450 pl, 150 Ill and 50 pl, respectively, of a 2% solution of Poncaux S (a red dye) where added to the above 3 suspensions and the suspensions were mixed until the dye was evenly dispersed (6) 40 pl droplets of each of the 3 suspensions were dispensed on to pre- prepared hydrophilic surfaces (parafilm), by pipette at room temperature (20-22-C).

(7) The hydrophobic surfaces were transferred into sealable boxes containing 500 grams of dried silica gel per 500 droplets and sealed.

(8) The boxes were placed in a refrigerator for 20 hours at 4 C. (9) The boxes were opened, the silica gel replaced by fresh material, and the boxes re-sealed and replaced in the refrigerator for a further 5 to 7 days.

(lo) The hydrophobic surfaces were removed from the sealed containers, and the dried droplets (now in the form of discs) were removed and stored in tubes containing freshly dried silica gel at -18 - C prior to use.

Example 3

E. cold NCTC 9001 was prepared and preserved in the form of discs using the process set out in Example 2 (51 aliquots of the BHI suspension were added to 16 ml aliquots of preserving medium, to give approximately 500 cfu per droplet before drying). After storage at 4-C for lo days, dried discs were re-constituted and the efficiency of plating was calculated by comparing replicate counts obtained from 5 discs directly plated onto Blood Agar and discs each plated on two selective media CLED and membrane lauryl sulphate agar (MLSA)). The results are shown in Table 1.

Table l

On Columbia Blood Agar, 5 replicates of five discs 136140 125 132 147132 148 140 114 147132 166 133 127140 153 125 121 145135 123 123 On CLED, 5 replicates of 5 discs 119 10897 121 110 106 123110 105 115 111128 117 107 126122 119 125 116113 108 115 C:: On MLSA, 5 replicates of 5 discs 87 8885 8391 97 80100 9375 96 8571 9086 77 8094 7981 82 10484 87102 The mean count per replicate is 135 cfu on Blood Agar, 115 cfu on CLED and 87 cfu on MLSA.

This gives an efficiency of plating relative to Blood Agar of for CLED and 64 % for MLSA.

Example 4

The organism set out below were all cultured and preserved using the materials and method set out in Examples 1 and 2.

After storage at 4 C for 4 days, 30 discs of each organism were reconstituted and counted. The mean count of the 30 replicates was calculated, together with the range and the 95 confidence limits, as set out in Table 2. These results indicate the generally high reproducibility of counts from different discs of drying materials.

Table 2

Strain NCTC Media Mean Range 95% CL B cereus 7464 BCM 31 25-38 17-49 B cereus 7464 BCM 35 27-45 20-54 B cereus 7464 BCM 36 27-46 21-55 B cereus 7464 BCM 33 19-44 19-52 E cold 12950 LSMB 59 48-70 39-83 E cold 12950 LSMB 59 44-75 39-83 E cold 12950 LSMB 58 40-69 38-82 E cold 12950 LSMB 59 40-79 39-83 Ent faecium 12952 S+B 98 78-128 72-128 Ent faecium 12952 S+B 97 79-110 71-127 Ent faecium 12952 S+B 99 80-139 73-129 Ps aeruginosa 12951 PsM 41 27-68 25-61 Ps aeruginosa 12951 PsM 41 32- 56 25-61 S poona 4840 BA 14 5-20 5-27 S enteritidis 6676 BA 9 3-20 2-20 S pullorum 10706 BA 12 10-19 6-19 S panama "wild" BA 6 1-10 1-16 S anatum "wild" BA 25 17-35 13-41 S dublin "wild" BA 26 16-37 13-43 S gold coast 13175 BA 11 3-23 BCM - Bacillus cereus medium (Oxoid) LSMB - Lauryl sulphate membrane broth (Oxoid) + B - Slanitz and Bartley medium (Oxoid) PsM - Pseudomonas medium (Oxoid) BA - Blood agar (Colombia Base (Oxoid) f-

Example 5

The organisms listed in Table 3 were all preserved using the materials and method set out in Examples 1 and 2, and comparative counts were made for the cultures before processing ("wet") and following reconstitution immediately after processing ("dryt') . Discs of each organism were then placed in glass tubes placed directly in to heating blocks at 45 and stored for 24 days (60 days for B. cereus). Discs were removed at 2, 5, 10 and 24 days, reconstituted and counted, and the results are set out in Table 3. In most cases it will be seen that good survival was obtained up to 24 days at 45 C. After 60 days storage the B. cereus count was 29 CFU and it is believed that the early drop in count was due to the death of vegetative cells leaving a more durable spore population.

Table 3

Stability on storage, Accelerated degeneration at 45 C, CFU per disc Wet Dry %Survival 2 5 10 24 days | Str pyogenes 8306 251 1 171 68 191 147 124 184 Ps aeruginosa 10662 134 91 68 26 7 2 0 Ent faecalis 775 131 161 100 191 121 11S 46 Staphylococcus 251 220 88 134 111 65 35 aureus 6571 List monocytogenes 213 211 99 131 64 72 14 B cereus 7464 54 44 81 35 21 20 26 S. gold coast 13175 193 213 100 144 188 155 135 it'

Example 6

Example 5 was repeated using S.gold coast NCTC 13175, Enterococcus faecalis NTCT 775, Staph.aureus NCTC 6571 and Legionella SP using discs containing approximately 104 cfu per disc. Discs were stored at 37 C for 252 days, reconstituted and counted. Compared to disc stared at -18 C, survival rates of 48%, 43%, 6% and 5%, respectively, were found.

Example 7

E. cold NCTC 9001 was prepared and preserved by the materials and methods of Examples 1 and 2. The counts of organisms dispersed in the preserving medium were adjusted so that one set of discs containing approximately 2000 CFU per disc and one set containing approximately 10000 CFU per disc were prepared. After storage at 4 C for 10 days, individual discs were reconstituted in brain heart infusion media. Discs nominally containing 2000 CFU were dispersed in 2ml of BHI and discs nominally containing 10000 CFU were dispersed in lOml BHI.

0.67 Al of each of the suspensions were spread onto agar plates comprising either blood agar using a Colombia base (BA), cystine lysine electrolyte depleted media (CLED) or membrane lauryl sulphate agar (MLSA). Incubation was carried out as shown in

table 4

Table 4

Run 1, one disc dissolved in 2 ml of BHI and plated onto BA 57 58 58 59 63 64 64 64 66 77 78 CLED 50 52 53 54 56 59 62 62 67 70 71 G=.

incubated at 37 - C Run 2, one disc dissolved in lOml of BHI and plated onto BA 53 59 61 62 63 66 69 72 73 75 76 78 CLED 50 50 51 53 54 54 59 59 61 62 63 67 incubated at 37 ' C Run 3, one disc dissolved in lOml of BHI and plated onto BA 55 59 60 61 61 66 66 67 67 69 70 86 CLED 50 50 51 51 51 56 56 58 63 67 74 incubated at 44 - C Run 4, one disc dissolved in lOml of BHI and plated onto BA 48 63 64 64 64 65 66 66 69 71 72 76 CLED 51 51 51 55 56 57 57 57 58 59 60 64 MSLA 37 38 41 41 42 43 44 45 47 47 52 54 incubated at 44 C

Example 8

Dried discs of Staph aureus, Strep. pyogenes, Enterococcus faecalis, Klebsiella aerogenes, Hemophilus influenzas, Listeria monocytogenes and B. cereus were prepared according to the procedures of Examples 1 and 2. The dried discs contained approximately 105 cfu's per disc. Dried droplets containing similar quantities of the above organisms were also prepared using preserving medium prepared according to the Examples of EP1003837 and WO01/05941. These suspensions were spotted out and left to dry at 4 C for one week.

Sample discs were transferred to a heating block set at 45 C and incubated for ten days.

Direct plating of these discs on support media showed a 2 to 3 loge or greater reduction in the viable count for discs prepared according to the Examples of EP1003837 and WO01/05941 compared to discs prepared by the methods of Examples 1 and 2. Apart from the H. influenzas, disc prepared by the methods of Examples 1 and 2 lost less than 1 logo of the original count.

Claims

1. A preserving medium for use in preserving biological material, comprising water, 5 to 30 wt% of one or more polyhydric or sugar alcohols and 5 to 30 wt% of one or more colloidal or soluble crystalloid materials, and being buffered to a pH of 6.5 to 7.5.

2. A preserving medium as claimed in claim 1, wherein the one or more polyhydric or sugar alcohols is selected from adonitol, sorbitol and mixtures thereof.

3. A preserving medium as claimed in claim 1 or claim 2, comprising 5 to 20 wt% of the one or more polyhydric or sugar alcohols.

4. A preserving medium as claimed in any preceding claim, wherein the one or more colloidal or soluble crystalloid materials is selected from bovine serum albumin, ovalbumin, carboxymethyl cellulose, gum arable, alginate, gelatin, DEAE, Ficol, polyvinylpyrrolidone and mixtures thereof.

5. A preserving medium as claimed in any preceding claim, comprising 5 to 20 wt% of the one or more colloidal or soluble crystalloid materials.

6. A preserving medium as claimed in any preceding claim, further comprising up to 15 wt% of a soluble peptide digest.

7. A preserving medium as claimed in any preceding claim, further comprising up to 12 wt% hydrolysed or partially hydrolyzed gelatin.

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8. A preserving medium as claimed in any preceding claim, having a viscosity of 1 to 10 mPa.s at 25 C and a viscosity of 500 to 1500 mPa.s at 4 C.

9. A method for preserving biological material comprising: (1) dispersing the biological material in a preserving medium as claimed in any preceding claim, and (2) drying the resulting dispersion above the triple point of the mixture, at a temperature of -5 C to 20 C.

10. A method as claimed in claim 9, wherein drops of the preserving medium containing biological material are dispensed onto a hydrophobic surface for drying.

11. A method as claimed in claim 10, wherein the dispensing is carried out at room temperature.

12. A method as claimed in claim 10 or claim 11, wherein Arm the hydrophobic surface is selected from parafilm; waxed paper; surfaces coated with paraffin wax, oil or Vaseline; silconised glass or plastics, such as polythene, polystyrene or teflon.

13. A method as claimed in any of claims 10 to 12, wherein the preserving material containing biological material is dispensed on to the hydrophobic surface in predetermined volumes.

14. A method as claimed in claim 13, wherein the predetermined volume is from 1 pi to 50 p1.

15. a method as claimed in any of claims 9 to 14, wherein the drying temperature is from 3 to 5 C.

16. A method as claimed in any of claims 9 to 15, wherein the drying is carried out in sealed containers, and means are provided for removing water from the atmosphere surrounding the materials to be dried.

17. A method as claimed in any of claims 9 to 16, wherein the drying is carried out until the moisture content is reduced to 6% wt% or less.

18. A preserving medium as herein defined.

19. A method of preserving biological material as herein defined.