GB1581718A - Stabilised leukocytes - Google Patents
Stabilised leukocytes Download PDFInfo
- Publication number
- GB1581718A GB1581718A GB12014/78A GB1201478A GB1581718A GB 1581718 A GB1581718 A GB 1581718A GB 12014/78 A GB12014/78 A GB 12014/78A GB 1201478 A GB1201478 A GB 1201478A GB 1581718 A GB1581718 A GB 1581718A
- Authority
- GB
- United Kingdom
- Prior art keywords
- leukocytes
- medium
- viability
- solution
- foetal calf
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
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- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 239000011775 sodium fluoride Substances 0.000 description 1
- 235000013024 sodium fluoride Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 229920001059 synthetic polymer Polymers 0.000 description 1
- 229960002898 threonine Drugs 0.000 description 1
- FGMPLJWBKKVCDB-UHFFFAOYSA-N trans-L-hydroxy-proline Natural products ON1CCCC1C(O)=O FGMPLJWBKKVCDB-UHFFFAOYSA-N 0.000 description 1
- 229960004799 tryptophan Drugs 0.000 description 1
- 231100000747 viability assay Toxicity 0.000 description 1
- 238000003026 viability measurement method Methods 0.000 description 1
- 229940011671 vitamin b6 Drugs 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/14—Blood; Artificial blood
- A61K35/15—Cells of the myeloid line, e.g. granulocytes, basophils, eosinophils, neutrophils, leucocytes, monocytes, macrophages or mast cells; Myeloid precursor cells; Antigen-presenting cells, e.g. dendritic cells
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Hematology (AREA)
- Immunology (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Cell Biology (AREA)
- Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Developmental Biology & Embryology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Dentistry (AREA)
- Wood Science & Technology (AREA)
- Environmental Sciences (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Description
(54) STABILISED LEUKOCYTES
(71) We, F. HOFFMANN-LA ROCHE & CO., Aktiengesellschaft, a Swiss Company of 124 - 184 Grenzacherstrasse, Basle, Switzerland, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement:
The present invention relates to a method for maintaining the viability of leukocytes or specific fractions of leukocytes.
Leukocytes are important for research and are used in various medical and medical diagnostic applications. Problems occur when it is necessary or desired to transport leukocytes from one location to another. Generally, leukocytes have hitherto been transported in the cold (e.g. at about 4"C) because they are unstable at higher temperatures such as room temperature unless used within 5 to 6 hours of their separation from whole blood.
Methods of collecting blood from donors or patients and separating leukocytes therefrom have hitherto been unsatisfactory for the formation of suspensions of leukocytes or specific fractions thereof which are stable at room temperature for extended periods. The usual method is to collect or place the blood in a syringe or container containing sufficient heparin to prevent clotting, to separate the leukocytes and to suspend them in a buffered medium.
Another method comprises collecting blood, defibrinating it with glass beads and separating the granulocytes using magnetic particles to leave the lymphocytes in the supernatant. Still another method comprises placing collected blood in Hank's solution containing sufficient heparin to provide 30 units/ml of blood. This latter method is about one-half as effective in maintaining viability compared to drawing blood directly into BSS-heparin (BSS = basic salt solution) or MEM-heparin (MEM = minimal essential medium) solution as in the present invention.
The foregoing methods and other known methods of separating leukocytes from blood do not result in a suspension of leukocytes which can be transported from one location to another at temperatures substantially above 4"C (e.g. room temperature). There is thus a need for a stable suspension of leukocytes which can be transported at temperatures which may vary from about 4"C to about 30"C and still remain viable for several days.
The prior art is illustrated by the following references:
Pattengale et al., The New England Journal of Medicine 291, No. 22, 1145-48, Nov. 28, 1974 describe the separation of leukocytes from heparinised blood, followed by lysis of the red cells and re-suspension of the leukocytes in a suitable medium containing 10% foetal bovine serum.
Schumm petal., Europ. J. Cancer 10, 107-113 (1974) also disclose the drawing of blood into heparinised vials, isolation of the leukocytes and suspension of the leukocytes in Hank's medium.
Zucker-Franklin et al., Proc. Nat. Acad. Sci. USA 71, No. 7, 2711-2714 describe the obtention of purified leukocytes from heparinised blood, the purified leukocytes then being suspended in a suitable medium containing 15% foetal calf serum.
Meckeretal., Oncology30, 177-191 (1974) describe a method wherein blood is collected in Hank's solution containing sufficient heparin to provide 30 units/ml of blood.
Chisarietal., The Journal of Experimental Medicine 142, 1092-1107(1975) describe the conventional technique of layering leukocytes on a Ficoll-Hypaque barrier. This reference also discloses the suspension of leukocytes in a suitable medium supplemented with antibiotics (i.e. penicillin and streptomycin).
Boyum, Scand. J. Clin. Lab. Invest. 21 (Suppl.) 97, 77 (1968) describes a method of isolating leukocytes wherein blood is collected in a heparinised syringe, diluted with phosphate buffer and centrifuged on a layer of Ficoll-Hypaque.
Chang et al., Biochemical and Biophysical Research Communications 64, No. 2, 539-545 (1975) is similar to the disclosure of Chisari in that it discloses blood being drawn into heparinised syringes and treated to purify the leukocytes which are then suspended in a .suitable medium containing 20% heat-inactivated foetal calf serum, penicillin and streptomycin.
West et al., Clinical Immunology and Immunopathology 5, 60-66 (1976) describe the purification of leukocytes according to the method of Boyum, supra, and the mixing of the leukocytes with heat-inactivated foetal calf serum.
Muijsson etal., Biochemical Genetics 13, Nos. 7/8, 501-509 (1975) describe the preparation of leukocytes by a method very similar to those described earlier, i.e. drawing blood into
heparinised saline, layering the leukocytes on Ficoll-Hypaque, centrifuging and resuspending the leukocytes in a suitable medium.
Vanky et al., J. Nat. Cancer Inst. 47, No. 1, 95-103 (July, 1971) describe a method of
defibrinating blood with glass beads and separating granulocytes by magnetic particles.
Lionetti et al., United States Patent Specification No. 4 004 975, describe a method of
preserving granulocytes which includes the isolation and freezing of the leukocytes.
None of the foregoing references describes nor suggests a means whereby leukocytes can
be maintained in a viable state for a period of several days at temperatures substantially in
excess of 4"C.
The present invention relates to a method for the production of a temperature-stable
leukocyte suspension and to the resulting suspension which is suitable for transportation from
one location to another at temperatures above 40C.
More particularly, the present invention is concerned with a method for maintaining the
viability of leukocytes, which method comprises (a) drawing blood from a patient or donor
directly into a mixture of an anticlotting agent and a first medium which sustains the viability
of leukocytes. (b) separating the leukocytes by centrifugation on to a solution having a
specific gravity of about the same as the leukocytes being separated containing a mixture of a
water-soluble synthetic copolymer of sucrose and epichlorohydrin with an average molecular
weight of 400,000 + 100,000 and sodium diatrizoate, and (c) collecting the leukocytes and
placing them in containers having a second medium which sustains the viability of the
leukocytes, said medium containing antibiotics and a substance selected from heat
inactivated foetal calf serum, protein substitutes equivalent to heat-activated foetal calf
serum and protein derivatives equivalent to heat-inactivated foetal calf serum.
The method provided by the present invention is generally applicable to all leukocytes (e.g.
macrophages, neutrophils, eosinophils, basophils, lymphocytes and the like). In a preferred
embodiment, the present method is applicable to lymphocytes.
The method provided by the present invention is carried out as follows:
(a) Blood is collected from a donor or patient directly into a syringe or other container
which contains an anticlotting agent and a medium which sustains the viability of the
leukocytes [i.e. a basic salt solution (BSS) which may or may not contain added amino acids
and vitamins or a minimal essential medium (MEM) and then mixed. It is important that the
blood be drawn directly into the BSS- or MEM-heparin mixture. Any other means of
combining the materials gives unsatisfactory results because the resulting leukocytes lose
viability. For example, the viability of lymphocytes in suspension produced according to the
method of this invention is about 90% to 100% for about a week at room temperature while
analogous processes result in about one-half the viability of lymphocytes.
Suitable anticlotting agents are ethylenediaminetetraacetic acid, sodium fluoride, sodium
citrate. heparin and the like, preferably heparin. The amount of anticlotting agent used can
vary with the volume of blood in the sample, but it should be sufficient to guarantee that no
clotting occurs. When heparin is used, for example, approximately 20 USP units of heparin
per ml of blood, depending on the specific heparin used, is sufficient.
The amount of BSS or MEM used should be sufficient to guarantee that the leukocytes in
the blood remain viable. About 30 %5 0 % by volume, based on the blood volume, of the BSS
or MEM is usually used. Various BSS or MEM formulations are suitable for use in the present
invention. Some of the common commercial BSS's and MEM's are as follows: Earle's MEM,
Hanks' MEM. RPMI medium, F-13 medium, Hanks' F-12 medium, Hanks' F-10 medium,
MB75211, McCoy 5a medium, Medium 499, Medium L-15, NCTC 109, Scherers Mainte
nance Solution. Earle's Basic Salt Solution, Hanks' Basic Salt Solution, Grey's Basic Salt
Solution.
Typical MEM and BSS solutions have the following formulations: Eagle Minimum Essen
tial Medium [Eagle, H. Science 130, 432 (1959)1.
Minimum Essential Medium (Eagle)
Components mg/litre
Amino acids
L-Arginine.HCl 126.4
L-Cystine 24.0 L-Glutamine 292.0
L-Histidine.HCl.H2 O 41.9
L-lsoleucine 52.5
L-Leucine 52.4 L-Lysine.HCl 73.1
L-Methionine 14.9 L-Phenylalanine 33.0
L-Threonine 47.6 L-Tryptophan 10.2
L-Tyrosine 36.2 L-Valine 46.8
Vitamins
D-Ca-Pantothenate 1.0
Choline chloride 1.0
Folio acid 1.0 i-Inositol 2.0 Nicotinamide 1.0
Pyridoxal.HCl 1.0
Riboflavin 0.1
Thiamine.HCl 1.0
Minimum Essential Medium (Eagle
Components mg/litre
Inorganic salts and other components
Earle's BSS
CaCl2.2H2O 265.0
KCI 400.0 MgSO4.7H2O 200.0
NaCI 6800.0
NaHCO3 2200.0 NaH2PO4.H2O 140.0
Dextrose 1000.0
Phenol red 10.0
Components
Hanks' BSS
CaCl2.2H2 0 186.0
KCI 400.0 KH2PO4 60.0 MgSO4.7H20 200.0
NaCI 8000.0
NaHCO3 350.0 Na2HPO4.7H20 90.0
Dextrose 1000.0
Phenol red 20.0 Minimum Essential Medium (Eagle)
Suspension Medium BSS mgllitre KCI 400.0
NaCI 6800.0
NaHCO3 2200.0 NaH2PO4.H20 1500.0
MaCl2.6H2O 200.0
Dextrose 1000.0
Phenol red 10.0
Components
Non-essential amino acids (NEAA) kAlanine 8.90 kAspartic acid 13.30
L-Asparagine 13.21 LGlutamic acid 14.70
Glycine 7.50
L-Proline 11.50
L-Serine 10.50
Earle's MEM is vialed at pH 7.0 to 7.2.
Hanks' MEM is vialed at pH 7.2 to 7.4.
RPMI Media 1640 [Moore et al]. JAMA 199, 519-524 (1967); In Vitro 6, No. 2 (1970)].
RPMI Media 1640
Components mg/litre
Amino acids
L-Alanine -- kArginine (free base) 200.0
L-Asparagine 50.0
L-Aspartic acid 20.0
L-Cystine 50.0
L-Glutarnic acid 20.0
L-Glutarnine 300.0
Glycine 10.0 L-Histidine (free base) 15.0 Hydroxy-L-proline 20.0 L-lsoleucine 50.0
L-Leucine 50.0 L-Lysine.HC1 40.0
L-Methionine 15.0
L-Phenylalanine 15.0 L-Proline 20.0
L-Serine 30.0
LThreonine 20.0
LTyrptophan 5.0 L-Tyrosine 20.0
L-Valine 20.0 RPMlMedia 1640 Components mg/litre
Vitamins
P-Aminobenzoic acid 1.00
Biotin 0.20
D-Ca-Pantothenate 0.25
Choline chloride 3.00
Folic acid 1.00
i-lnositol 35.00
Nicotinamide 1.00
Pyridoxine.HCl 1.00
Riboflavin 0.20
Thiamine.HCl 1.00
Vitamin Bl2 0.005
Other components
Dextrose 2000.0 Glutathione (reduced) 1.0
Phenol red 5.0
Inorganic salts
Ca(NO3)24H2O 100.0
KC1 400.0 MgSo4-7H20 100.0
NaCI 6000.0
NaHCO3 2000.0 Na2HPO4,7H2O 1512.0
vialed at pH
7.2 to 7.4 Earle's, Grey's and Hanks' Balanced Salt Solution [Earle, J. Nat. Cancer Inst. 4, 165-212 (1943); Geyetal.. Am. J. Cancer 27,45-76(1936); Hanksetal., Proc. Soc. Exp. Biol. and Med. 71. 196-200 (1949)].
lGalanced Salt Solutions
Components Earle's Gey's Hanks'
(mg/litre) (mgllitrel (mgllitrel NaC1 6800 8000 8000 KCI ' 400 375 400 CaCl2'2H2O 265 225 186 'Mg"SO47H2O 200 70 200 MgG-26H2O -- 210 - NaH2 PO4 H2 140 -- - Na2HPO47H2O -- 226 90 KH2 PO4 -- 30 60
Dextrose 1000 1000 1000
Phenol red 10 -- 20 NaHCO 3 2200 227 350
These solutions are available from Microbiological Associates, Bethesda, Maryland.
Step (a) described herein makes it possible to carry out the remaining steps of the present method to achieve the stable leukocyte composition. After mixing the ingredients together the following steps are carried out.
(b) The blood-heparin-MEM or -BSS mixture is layered on a Ficoll-Hypaque solution in a glass container, preferably a test tube. This is carried out by carefully pouring the mixture on to the solution in the container. The volume ratio of the Ficoll-Hypaque mixture to the blood is from 1:1 to 1:2, preferably 1:1. The relative amounts of the mixture and solution can vary.
However, slightly more volume of mixture is usually used. The amounts of Ficoll and
Hypaque can vary. However, the relative amounts should be such that the specific gravity of the Ficoll-Hypaque solution is about the same as that of the particular leukocytes being separated. For example, when lymphocytes are separated, the specific gravity should be about 1.07 to 1.08. Ficoll is a synthetic high polymer made by the copolymerisation of sucrose and epichlorohydrin, the average molecular weight being 400,000 + 100,000. Hypaque is sodium diatrizoate. Ficoll and Hypaque are both soluble in water. Distilled and/or deionised water is used to form the Ficoll-Hypaque solution used in the present method. Ficoll is available from Pharmacia, Piscataway, New Jersey. Hypaque is available from Winthrop
Laboratories, New York City.
(c) The blood-heparin-BSS or -MEM mixture layered on the Ficoll-Hypaque solution is centrifuged at 40C to separate the leukocytes from the plasma. The centrifugation is carried out in the cold to guarantee that there is no deterioration of the leukocytes from heat caused by the centrifugation forces. The revolutions per minute of the centrifuge should be such that about 400 times gravity results for a sufficient time to allow the leukocytes to layer on the
Ficoll-Hypaque solution (e.g: about 30 to 40 minutes).
(d) The resulting supernatant plasma from step (c) is then decanted off and the leukocyte-containing layer is removed by conventional means (e.g. with a syringe). The leukocyte-containing layer is then mixed with BSS or MEM and centrifuged at about 400 times gravity for a time sufficient to allow the leukocytes to separate from the BSS or MEM (e.g. about 10 to 30 minutes). The supernatant is then decanted off. The pellet which remains contains the leukocytes.
The leukocyte pellet is suspended in BSS or MEM and an aliquot is taken so that the number of leukocyte cells can be counted. Any convenient means for counting the cells can be used. However. it has been found that a Coulter Counter (Coulter Electronics Inc., Hialeah,
Florida) is preferred. In addition, another aliquot is used to assay for the viability of the cells.
The conventional trypan blue dye exclusion method is used. The cell counting and viability assay are not part of the method provided by the present invention, but are used in commercial processes for quality control.
(e) After counting the cells and determining that they are viable, the BSS or MEM suspension is diluted with additional BSS or MEM suspension containing antibiotics and heat-inactivated foetal calf serum or a serum substitute equivalent thereto to achieve a final viable leukocyte concentration of about 0.5 x 106 viable cells per ml.
The antibiotics are added to guarantee the sterility of the leukocyte preparation. Uusually, groups of antibiotics having varied specific activities are added to achieve a broad spectrum of activity. The amounts of given antibiotics used will vary with their effective concentration as is well recognised. For example, a preferred combination of antibiotics is penicillin, streptomycin and gentamycin. When this preferred group of antibiotics is used, the diluting medium contains an amount sufficient to provide concentrations of 75 units,50,ugrams and 50 ,grams per ml, respectively, in the final preparation.
The heat-inactivated foetal calf serum or serum substitute equivalent thereto is used to maintain the leukocytes in the proper environment for viability. By this is meant that they provide protein in the media which acts to prevent loss of protein from the cell membranes.
Also, the presence of the source of protein in the environment acts in a sense to nourish the cell membrane.
The use of heat-inactivated foetal calf serum as an adjunct to the maintenance of various types of cells is well documented in the literature [see, for example, McCall et al., Ann.
Rheum. Dis., 25, pages 42-48 (1966); McLeod et al., Blood, Vol. 44, No. 4, pages 517-534;
Kovacs et al., J. of Inv. Dermatology, Vol. 63, pages 456-60 (1974); and Frabrikant et al.,
Radiology, Vol. 92, pages 1309-20 (1962), as well as the Zucker-Franklin etal and Changet al references referred to previously]. Serum substitutes which are recognised as being equivalent to heat-inactivated foetal calf serum are basically of two types, namely protein and synthetic media. Examples of protein substitutes include human serum albumin and autologous serum. Examples of synthetic media include serum-free culture systems containing as protective agents synthetic polymers such as methyl cellulose, sodium carboxymethyl cellulose, dextans, hydroxymethyl starch and the like. A detailed discussion of such serum substitutes is given by Taylor, in Journal of the National Cancer Institute, Vol. 53, No. 5,
November, 1974, pages 1449-1457 and in the references cited therein. Generally, the diluting medium (i.e. BSS or MEM) will contain from about 1% to about 10% by volume heat-inactivated foetal calf serum or a serum substitute equivalent thereto.
(f) The suspension from step (e), maintained at room temperature, is then transferred into sterile containers (e.g. glass, plastic and the like) for transportation. Each container should contain a convenient amount of leukocyte cells which retain substantially all their viability (i.e. about 2 to 10 x 106 leukocyte cells). The containers are packed into insulated containers and are ready for transportation. A preferred container is made from Styrofoam (Trade Mark covering foamed polystyrene).
When prepared as described, about 90% and more of the leukocyte cells in the suspension are stable for about a week.
Example
1 ml of heparin and 11 ml of F-13 medium (an MEM medium) are drawn into 9 30 ml syringe. The syringe needles are changed and 18 ml of blood from a donor are drawn directly into the syringe. The blood and heparin F- 13 media are mixed by inverting the syringe once or twice.
About 8 ml of the resulting mixture are layered on 6 ml of Ficoll-Hypaque solution in a 16 x 25 mm test tube and centrifuged at 1000 rpm (400 times gravity) at 40C for 37 minutes. The resulting supernatant plasma layer is decanted off. The lymphocyte rich layer is removed and transferred to 10 ml of F-13 media. The resulting mixture is centrifuged at 1000 rpm (400 times gravity) at 4"C for 10 minutes. The supernatant is removed to leave a pellet containing lymphocytes. 4.5 ml of F-13 media is added to the pellet and mixed well. A 0.1 ml aliquot is removed and put into 20 ml of counting solution (an isotonic solution) for a Coulter count (cell count on a Coulter Counter). Another aliquot is removed and the lymphocyte cells therein are assayed for viability using trypan blue dye exclusion method.
The suspension of lymphocytes in F- 13 media is diluted to 0.5 x 106 viable cells per ml with F-13 media containing 1000 units/ml penicillin, 50 g/ml streptomycin and 10% by volume heat inactivated foetal calf serum. The resulting samples, maintained at room temperature, are transferred into sterile glass containers so that each glass container has 5 x 106 lymphocyte cells. The glass containers are packed into Styrofoam containers and are ready for shipment, e.g. by air freight. The viability of lymphocyte cells in suspension prepared according to this example and shipped by air freight was as follows:
The following Example illustrates the present invention:
Example
Table
:'Sample Age of cells (days) % Viability 1 4 97 2 4 98 3 5 94 4 3 92
5 1 93 6 4 91
The data in the foregoing Table demonstrates the effectiveness of the method provided by this invention.
The Ficoll-Hypaque solution used in this Example is prepared according to the following directions:
A. Preparation of Ficoll:
1. Weigh 18 grams of Ficoll.
2. Add sufficient distilled water to the Ficoll to total 200 ml.
3. Mix until all has dissolved.
4. Mix for 5 minutes with a magnetic stirrer.
B. Preparation of Hypaque:
1. Use two 25 ml burettes; one for distilled water and the other for a 50% aqueous
Hypaque solution.
2. Dilute 56.5 ml of the 50% Hypaque solution with 26.84 ml of distilled water.
C. Preparation of Ficoll-Hypaque:
1. Mix all of the resulting Hypaque solution with all of the resulting Ficoll solution.
2. Fill a graduated cylinder with 250 ml of the Ficoll-Hypaque solution.
3. Measure the specific gravity of the solution with a hydrometer; if the specific gravity is less than 1.07-1.08 add more Hypaque solution, if it is greater than 1.08 add distilled water.
4. Autoclave the solution.
In addition to maintaining the viability of, for example, lymphocytes, the method provided by the present invention permits the recovery of over 90 % of the total cells (e.g. lymphocytes) present. This is due to the fact that the medium mixed with the anticlotting agent helps prevent clumping of the cells.
WHAT WE CLAIM IS:
1) A method for maintaining the viability of leukocytes, which method comprises (a) drawing blood from a patient or donor directly into a mixture of an anticlotting agent and a first medium which sustains the viability of leukocytes, (b) separating the leukocytes by centrifugation on to a solution having a specific gravity of about the same as the leukocytes being separated containing a mixture of water-soluble synthetic copolymer of sucrose and epichlorohydrin with an average molecular weight of 400,000 + 100,000 and sodium diatrizoate, and (c) collecting the leukocytes and placing them in containers having a second medium which sustains the viability of the leukocytes, said medium containing antibiotics and a substance selected from heat-inactivated foetal calf serum and protein derivatives equivalent to heat-inactivated foetal calf serum.
2) A method according to claim 1, wherein the anticlotting agent is heparin.
3) A method according to claim 1 or claim 2, wherein the first medium which sustains the viability of leukocytes is a basic salt solution.
4) A method according to claim 1 or claim 2, wherein the first medium which sustains the viability of leukocytes is a minimum essential medium.
5) A method according to claim 1 or claim 2, wherein the second medium which sustains the viability of the leukocytes is a basic salt solution containing antibiotics and a substance selected from heat-inactivated foetal calf serum, protein substitutes equivalent to heat inactivated foetal calf serum and protein derivatives equivalent to heat-inactivated foetal calf serum.
6) A method according to claim 1 or claim 2, wherein the second medium which sustains the viability of leukocytes is a minimum essential medium containing antibiotics and a substance selected from heat-inactivated foetal calf serum, protein substitutes equivalent to heat-inactivated foetal calf serum and protein derivatives equivalent to heat-inactivated foetal calf serum.
**WARNING** end of DESC field may overlap start of CLMS **.
Claims (4)
- **WARNING** start of CLMS field may overlap end of DESC **.Example Table :'Sample Age of cells (days) % Viability 1 4 972 4 983 5 94 4 3 925 1 936 4 91 The data in the foregoing Table demonstrates the effectiveness of the method provided by this invention.The Ficoll-Hypaque solution used in this Example is prepared according to the following directions: A. Preparation of Ficoll: 1. Weigh 18 grams of Ficoll.
- 2. Add sufficient distilled water to the Ficoll to total 200 ml.
- 3. Mix until all has dissolved.
- 4. Autoclave the solution.In addition to maintaining the viability of, for example, lymphocytes, the method provided by the present invention permits the recovery of over 90 % of the total cells (e.g. lymphocytes) present. This is due to the fact that the medium mixed with the anticlotting agent helps prevent clumping of the cells.WHAT WE CLAIM IS: 1) A method for maintaining the viability of leukocytes, which method comprises (a) drawing blood from a patient or donor directly into a mixture of an anticlotting agent and a first medium which sustains the viability of leukocytes, (b) separating the leukocytes by centrifugation on to a solution having a specific gravity of about the same as the leukocytes being separated containing a mixture of water-soluble synthetic copolymer of sucrose and epichlorohydrin with an average molecular weight of 400,000 + 100,000 and sodium diatrizoate, and (c) collecting the leukocytes and placing them in containers having a second medium which sustains the viability of the leukocytes, said medium containing antibiotics and a substance selected from heat-inactivated foetal calf serum and protein derivatives equivalent to heat-inactivated foetal calf serum.2) A method according to claim 1, wherein the anticlotting agent is heparin.3) A method according to claim 1 or claim 2, wherein the first medium which sustains the viability of leukocytes is a basic salt solution.4) A method according to claim 1 or claim 2, wherein the first medium which sustains the viability of leukocytes is a minimum essential medium.5) A method according to claim 1 or claim 2, wherein the second medium which sustains the viability of the leukocytes is a basic salt solution containing antibiotics and a substance selected from heat-inactivated foetal calf serum, protein substitutes equivalent to heat inactivated foetal calf serum and protein derivatives equivalent to heat-inactivated foetal calf serum.6) A method according to claim 1 or claim 2, wherein the second medium which sustains the viability of leukocytes is a minimum essential medium containing antibiotics and a substance selected from heat-inactivated foetal calf serum, protein substitutes equivalent to heat-inactivated foetal calf serum and protein derivatives equivalent to heat-inactivated foetal calf serum.7) A method according to any one of claims 1 to 6 inclusive, wherein the leukocytes arelymphocytes and the specific gravity of the solution in step (b) is 1.07 to 1.08.8) A suspension of leukocytes containing 90% to 100% viable leukocytes in a medium which sustains the viability of leukocytes, said medium containing antibiotics and a substance selected from heat-inactivated foetal calf serum, protein substitutes equivalent to heatinactivated foetal calf serurn and protein derivatives equivalent to heat-inactivated foetal calf serum.9) A suspension according to claim 8, wherein the leukocytes are lymphocytes.10) A suspension according to claim 8 or claim 9, wherein the medium which sustains the viability of leukocytes is a minimum essential medium.I I ) A suspension according to claim 8 or claim 9, wherein the medium which sustains the viability of leukocytes is a basic salt solution.12) A suspension according to any one of claims 8 to 11 inclusive which contains heat-inactivated foetal calf serum.4. Mix for 5 minutes with a magnetic stirrer.B. Preparation of Hypaque: 1. Use two 25 ml burettes; one for distilled water and the other for a 50% aqueous Hypaque solution.2. Dilute 56.5 ml of the 50% Hypaque solution with 26.84 ml of distilled water.C. Preparation of Ficoll-Hypaque: 1. Mix all of the resulting Hypaque solution with all of the resulting Ficoll solution.2. Fill a graduated cylinder with 250 ml of the Ficoll-Hypaque solution.3. Measure the specific gravity of the solution with a hydrometer; if the specific gravity is less than 1.07-1.08 add more Hypaque solution, if it is greater than 1.08 add distilled water.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US78259777A | 1977-03-29 | 1977-03-29 | |
| US05/858,129 US4152208A (en) | 1977-03-29 | 1977-12-06 | Stabilized leucocytes |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| GB1581718A true GB1581718A (en) | 1980-12-17 |
Family
ID=27120014
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| GB12014/78A Expired GB1581718A (en) | 1977-03-29 | 1978-03-28 | Stabilised leukocytes |
Country Status (6)
| Country | Link |
|---|---|
| JP (1) | JPS53122283A (en) |
| AT (2) | ATA287878A (en) |
| DE (1) | DE2813548A1 (en) |
| DK (1) | DK136278A (en) |
| FR (1) | FR2385400A1 (en) |
| GB (1) | GB1581718A (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3009126A1 (en) * | 1980-03-10 | 1981-10-08 | Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V., 3400 Göttingen | METHOD FOR OBTAINING FUNCTIONAL AND MORPHOLOGICALLY INTACT AND VIABLE LEUCOCYTES FROM BLOOD |
-
1978
- 1978-03-28 DK DK136278A patent/DK136278A/en not_active IP Right Cessation
- 1978-03-28 FR FR7808865A patent/FR2385400A1/en not_active Withdrawn
- 1978-03-28 GB GB12014/78A patent/GB1581718A/en not_active Expired
- 1978-03-28 AT AT287878A patent/ATA287878A/en unknown
- 1978-03-28 JP JP3494378A patent/JPS53122283A/en active Pending
- 1978-03-28 AT AT0217878A patent/AT364085B/en not_active IP Right Cessation
- 1978-03-29 DE DE19782813548 patent/DE2813548A1/en not_active Withdrawn
Also Published As
| Publication number | Publication date |
|---|---|
| ATA287878A (en) | 1981-02-15 |
| DE2813548A1 (en) | 1978-10-05 |
| JPS53122283A (en) | 1978-10-25 |
| AT364085B (en) | 1981-09-25 |
| FR2385400A1 (en) | 1978-10-27 |
| DK136278A (en) | 1978-09-30 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PS | Patent sealed [section 19, patents act 1949] | ||
| PCNP | Patent ceased through non-payment of renewal fee |