WO1998035021A1 - Preparation de spheroides et utilisation de ces derniers pour des applications therapeutiques ou diagnostiques - Google Patents

Preparation de spheroides et utilisation de ces derniers pour des applications therapeutiques ou diagnostiques Download PDF

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WO1998035021A1
WO1998035021A1 PCT/GB1998/000343 GB9800343W WO9835021A1 WO 1998035021 A1 WO1998035021 A1 WO 1998035021A1 GB 9800343 W GB9800343 W GB 9800343W WO 9835021 A1 WO9835021 A1 WO 9835021A1
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spheroids
spheroid
cells
cryopreserved
cryopreservation
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PCT/GB1998/000343
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English (en)
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Christopher Kenneth Atterwill
Wendy Maria Purcell
Jinsheng Xu
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University Of Hertfordshire
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Priority claimed from GBGB9702335.2A external-priority patent/GB9702335D0/en
Priority claimed from GBGB9709899.0A external-priority patent/GB9709899D0/en
Application filed by University Of Hertfordshire filed Critical University Of Hertfordshire
Priority to AU58755/98A priority Critical patent/AU5875598A/en
Publication of WO1998035021A1 publication Critical patent/WO1998035021A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/067—Hepatocytes
    • C12N5/0671—Three-dimensional culture, tissue culture or organ culture; Encapsulated cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/20—Heating or cooling
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/14—Coculture with; Conditioned medium produced by hepatocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2503/00—Use of cells in diagnostics

Definitions

  • the present invention relates to artificially produced aggregates of cells in the form of three dimensional structures which are known as spheroids.
  • Spheroids appear to restrict cellular division whilst enhancing biochemical and morphological differentiation (see Seeds et al., 1980) thereby more closely reflecting the in vivo development process compared with primary monolayer cultures. Spheroids are therefore suitable for long term culturing experiments and in the presence of Vitamin E can remain viable for up to two months (Halks-Miller et al., 1982).
  • Spheroidal cultures were initially grown from limb buds, liver and kidney tissue (Moscona, 1961) succeeded by whole brain and retinal spheroids (Garber and Moscona, 1972).
  • brain reaggregate cultures have been studied extensively because of the lack of complex CNS cultures that can be maintained long term in vitro.
  • the yield of tissue is sufficient for standard biochemical analysis of whole spheroids as well as subcellular fractions (Trapp and Richelson, 1980). Under certain circumstances the cells can migrate and organize themselves into aggregates which resemble the architecture of the brain area sampled.
  • spheroids can be produced which histotypically resemble a particular brain region so long as such discrete areas or nuclei have been used as the starting material (DeLong, 1970; Garber, 1977; Levitt et al., 1976; Tsai, 1976). This is useful for closer examination of selective CNS toxicity caused by specific agents for example, MPTP.
  • CNS spheroids comprise an integrated population of neurones, astrocytes and other glial cell types for example, oligodendrocytes. Spheroids can therefore be sampled at various developmental stages as the CNS cells develop from undifferentiated neuroepithelial cells to a population of mature neurones, astrocytes and oligodendrocytes, correlating with in situ CNS development (Seeds et al. , 1980).
  • fetal cells in a dissociated cell suspension, first form a group of randomly associated cells on rotation, which then migrate to produce an organised spheroid.
  • astrocytes form a shell or 'glia limitans' around the outside of the structure with neurones and oligodendrocytes arranged within the interior.
  • Smaller neurones such as the granule cells are located innermost whilst larger neurones (including Purkinje cells) are found near the surface of the spheroid as in mature tissue in situ (Seeds et al. , 1980).
  • An in depth developmental description of brain spheroids is provided by Goldsmith and Berens (1990).
  • Neurotrophic viral infections have been studied in brain spheroids (Pulliam et al. , 1984) as well as myelination (Matthieu et al., 1979; Louglin et al., 1994) and the development of antioxidant enzyme systems (Aspberg and Tottmar, 1992) as well as pure developmental studies (Honegger and Schilter, 1992). More recently, brain spheroids have been used as a source of tissue for transplantation studies (Marienhagen et al., 1994).
  • liver spheroids Honegger & Schilter (1992) first noted that it was possible to culture hepatic (liver) spheroids from foetal tissue and then coculture them with adult rat brain spheroids. This is particularly useful for neurotoxicants and compounds requiring metabolic activation and avoids the inherent toxicity of liver microsomal S9 fractions to neural cultures. Their initial studies demonstrated that Phenobarbital- and 3-methylcholanthrene-exposed liver spheroids were able to produce neurotoxic metabolites of cyclophosphamide in vitro.
  • liver spheroids Since then, interest in liver spheroids has arisen for hepatoxicological test models in their own right (Ueno et al, 1992; George et al, 1996).
  • a method combining orbital rotation with substratum-coated plastic with PHEMA (2-hydroxyethyl methacrylate) allows liver spheroid culture from adult rat liver where the intraspheroidal hepatocytes retain many of the in vivo morphological characteristics (George et al, 1996).
  • Rat adult liver and foetal brain spheroids have now been co-maintained in the brain spheroid-based medium following separate initial cultivation in their optimal growth media in both our laboratory and that of Honegger & Schilter (1992).
  • the two spheroidal subtypes co-exist for around a week without significant loss of structure or increase a basal toxicological indicators. It is anticipated that such methodology will further progress in vitro neurotoxicity testing strategies for compounds such as Ecstasy (MDMA) which require hepatic metabolic activation before neurotoxic effects.
  • MDMA Ecstasy
  • spheroids are useful for many purposes. However their utility has been limited because they take a long time to prepare and typically only remain viable in culture for around 1-2 months.
  • cryopreservation of a limited number of cell types is already known, there is no disclosure in the literature of the successful cryopreservation of spheroids prior to the present invention.
  • spheroids In order to function as useful in vitro models of tissues/organs, spheroids should substantially retain their general three-dimensional structures. Furthermore the cells of the spheroids should retain viability and should also retain any biological characteristics which may be desired for spheroids to function as models of in vivo systems. A skilled person would realise that extreme techniques such as cryopreservation can disrupt individual cells and can also disrupt intercellular interactions which are needed to maintain a spheroids three-dimensional structure.
  • the electrolyte concentration inside and outside the cells can increase by several orders of magnitude relative to isotonic conditions, concentrated organic solvents in the freezing media permeate the cells, ice crystals intercalate the tissue and mechanically deform cells, and ice may form inside cells, disrupting intracellular structures or between cells, disrupting cell-cell aggregation.
  • cryopreservation an approach based on the principle that chemical, biological and physical processes are effectively 'suspended' at cryogenic temperatures.
  • Spheroid cultures in particular present certain inherent difficulties with respect of developing a suitable cryopreservation protocol. Given their three-dimensional shape and size (typical range 100-1000 ⁇ m) it may be anticipated that surface cells may be exposed to toxic concentrations of cryoprotectant in order to attain the minimal necessary cryoprotectant concentration in the spheroid interior. Conversely, if care is taken not to harm the outer cells during cryopreservation, interior cells may have cryoprotectant concentrations too low to protect against freezing.
  • the present invention therefore represents a significant advance over prior art, in that the cryopreservation protocols for spheroid cultures allow an even distribution of cryoprotectant across a spheroid to be maintained such that no region is significantly damaged by over- or under-exposure to the cryoprotectant.
  • Cells for use in producing cryopreserved spheroids can be derived from any suitable tissue source, including infected tissue (as will be discussed later).
  • tissue source including infected tissue (as will be discussed later).
  • spheroids comprising neuronal cells e.g. brain spheroids
  • foetal tissue is preferred.
  • tissue from both foetal and non- foetal e.g. adult sources
  • Liver cells are particularly useful since they can be used to produce spheroids which have some of the characteristics of the liver and can therefore be used to model the in vivo metabolism of substances by the liver. This is useful, for example, in determining whether or not particular substances are likely to be toxic following metabolism by the liver.
  • spheroids derived from liver cells are co- cultured with spheroids comprising neurons (e.g. brain spheroids).
  • neurons e.g. brain spheroids
  • Spheroids can in principle be produced from any desired tissue or organ from any animal by disrupting a sample of the tissue or organ, preferably to individual cells or to small groups of cells (e.g. by mechanical disruption via gentle trituration through a Pasteur pipette) and causing aggregation of the cells by orbital rotation. They can be used as in vitro models and are therefore advantageous alternatives to using live animals.
  • Preferred models are mammalian (e.g. human, non-human primate, porcine, rodent) or avian (e.g. chick) models.
  • the present invention allows the use of spheroids to become much more widespread because they can be prepared at one site in large numbers using the information provided herein and can then be cryopreserved and transported in the cryopreserved state over large distances and for long time periods to other sites (e.g. to hospitals or laboratories which can even be located overseas). They can then be stored, cultured with ease and used when desired, thereby avoiding the need to prepare spheroids in situ. These advantages are especially important in respect of human tissues.
  • cryopreservation can allow spheroids to be preserved in a viable state for a long time until needed. It is anticipated that, like other cryopreserved cells and tissues, spheroids may be held in the cryopreserved state for a month or more without significant loss of functionality upon thaw. Indeed it is anticipated that they may be held for a year or more.
  • the present invention represents a significant technical development likely to be of major significance to the pharmaceutical, chemical and agricultural industries. It is particularly useful in the healthcare sector (e.g. in transplantation for drug/chemical testing and for screening purposes).
  • the spheroids comprise lesions.
  • Lesions can be provided (for example) by ibotenic acid or ethylcholine mustard aziridinium ion (ECMA) treatment of spheroids for cholinergic neurones (Pillar et al, 1993), MPTP or 6-hydroxydopamine (6-OHDA) for Dopaminergic or Dopaminergic/noradrenergic neurones respectively, Kainic acid, ecstasy (MDMA) or NMD A for excitotoxic lesions, iron (Fe) for free-radical/ oxidative stress- induced damage, 5,6-dihydroxy tryptamine for serotonergic neurones, in vitro ischaemic or anoxic manipulation to emulate stroke-induced damage, and aluminium or /3-amyloid ( ⁇ -AP) for Alzheimer-like neuropathology.
  • ECMA ethylcholine mustard aziridinium ion
  • NCE's novel therapeutic drugs/molecules
  • neurodegenerative conditions such as Alzheimer's Disease, Parkinson's Diseases, Stroke and lesions induced by pesticide, environmental/industrial chemical, or therapeutic (e.g. antibiotics) or recreational drug exposure, or through genetic predisposition (e.g. diabetic neuropathy).
  • therapeutic neurotrophic factors such as NGF, BDNF, GDNF etc since we have already shown e.g. that NGF treatment in vitro of rat brain spheroids can reverse ECMA- induced cholinergic damage (Atterwill & Meakin, 1990).
  • the lesioned spheroid model could also be used to investigate the molecular and cellular neuropathological processes accompanying such lesions for comparison to the pathophysiological mechanisms in animal models and humans in neurodegenerative states.
  • the spheroids can contain 'foreign' genes. These can be introduced by standard molecular biological techniques for cell transfection, for example, electroporation.
  • the appropriate reporter elements of promotor-reporter constructs are e.g. Chloramphenicol Acetyltransferase (CAT), Luciferase and Secreted Alkaline Phosphatase (SAP). These reporters can be used to detect activation of early response genes relevant to pharmacotoxicological applications (e.g. p53, bcl, c-fos, c-jun, NF- ⁇ B, etc).
  • spheroids are co-cultured with cells derived from blood vessels prior to cryopreservation (e.g. from capilliaries). This is useful in providing in vitro models of in vivo systems in which blood vessels are involved e.g. of the blood brain barrier (BBB).
  • BBB blood brain barrier
  • spheroids can be prepared from cells or tissues infected in vivo, ex vivo, or in vitro with microbiological organisms (e.g. viruses, bacteria, protozoa, etc.). Such 'infected' spheroids may be used to, for example, assess the efficacy of drugs on infective organisms, screening of novel anti-infective agents, follow the pathogenesis of infection, etc.
  • microbiological organisms e.g. viruses, bacteria, protozoa, etc.
  • EC endothelial cells
  • neural tissue cultures in the form of spheroids.
  • spheroids These organotypic cultures comprise neural and glial elements which may provide a suitable matrix for EC attachment and growth hence producing an in vitro BBB.
  • Whole rat brain spheroids have been shown to behave organotypically in toxicity studies (Atterwill et al, Br. J. Pharmacol. 83:89-102 (1984)). It has already been demonstrated that EC attach to reaggregates (Bystry et al, Hum. Expt. Tox. 2:171 (1996)).
  • Brain spheroids were prepared by the method of Atterwill et al (supra).
  • EC were prepared by the method of Abbott et al (J. Cell Sci. 103:23-27 (1992)).
  • RBE4 cells were cultured by the method of Roux et al (J. Cell Physiol. 159:101-113
  • Confluent cultures EC were stained at 5 days in vitro (DIV) and RBE4 were stained at 70% confluence with the fluorophore l,l'-Dioctadecyl-3,3,3',3'- tetramethylindodicarbocyanine perchlorate, (Dil), a non specific cell dye for two days. Dil was used to visualise EC and RBE4 cell attachment to the reaggregates. -lilt is important to note that the cryopreservation of spheroids is not only useful in respect of spheroids intended as animal models (e.g. of human or other mammalian systems). It is also useful in respect of spheroids which can themselves be used in treatment.
  • spheroids can be useful in providing bioartificial systems.
  • bioartificial liver BAL
  • Porcine hepatocytes may be suitable for use in bioartificial liver (BAL) systems (Saki et al, 1996). Such systems can provide effective treatment of patients with acute liver failure or can provide life-prolonging systems for patients awaiting orthotropic liver transplantation. Porcine hepatocytes express high hepatic functions in vitro and resemble human hepatocytes in morphology and functions.
  • BAL bioartificial liver
  • porcine hepatocytes have been reorganized into spheroids having a tissue-like structure in suspension culture, and they express higher hepatic functions in vitro as compared to conventional monolayers, as confirmed in the case of rat hepatocyte culture.
  • Spheroids can also be used to provide implants to be transplanted into and maintained in patients. They are therefore useful in restorative transplantation.
  • degenerative diseases or disorders are particularly useful in treating degenerative diseases or disorders since they can allow damaged cells or tissues or organs to be replaced.
  • the degenerative diseases or disorders may be genetically-based or may be caused by environmental factors (e.g. by exposure to toxic drugs or other chemicals).
  • neurodegenerative diseases e.g. Parkinson's disease, Alzheimer's disease or stroke
  • Neuronal degeneration caused by Ecstasy or by other drugs may also be treated.
  • Liver disease or damage may also be treated (e.g. by using bioartificial livers as described above or by providing implants).
  • the present invention allows such access to be achievable since large numbers of spheroids can be stored in a cryopreserved state and can be thawed and prepared for use shortly before being required.
  • cryopreservants can be used, although DMSO is preferred.
  • cryopreservants include permeating cryoprotectants besides DMSO can however be used: e.g. glycerol, 1,2-propanediol, acetamide, ethylclycol and propylene glycol (Karlsson & Mehmet, 1996; Chao et al, 1994).
  • Non-permeating cryopreservants may even be used: e.g. methy .cellulose poly vinyl pyrollidone, hydroxy ethyl starch and various sugar-based cryopreservants.
  • cryopreservant is at a level of at least 5% v/v (e.g. from 10-20% v/v) with respect to a composition comprising the spheroids and the cryopreservant immediately prior to cryopreservation.
  • spheroids are cooled and then maintained within a given temperature range before being cooled further. Without being bound by theory, it is possible that this procedure may give the cells constituting a spheroid sufficient time to acclimatize to cold temperatures and thereby to avoid cell death or damage which might otherwise occur.
  • the spheroids can be cooled to a temperature of from 1 to 8°C (e.g. from 2 to 6°C and preferably of about 4°C) and held for a period before further cooling. This period may be a period of at least 10 mins, preferably a period of at least 30 mins and typically a period of 40-60 mins. Such cooling can be conveniently achieved by using a laboratory refrigerator.
  • the spheroids may then be cooled to a temperature of from 0 to -50 °C (e.g. from -10 to -30°C, preferably of from -15 to -25°C) and held for a period before further cooling. This can be conveniently achieved by using a laboratory freezer. This period may be a period of at least 30 mins and is preferably of at least 1 hour
  • the final step in the cooling procedure will usually be a rapid cooling to a temperature of below -100°C e.g. of below -190°C. This can be done using liquid nitrogen as a coolant, into which is placed a resilient container containing the spheroids and the cryopreservant.
  • a further preferred feature of methods of the present invention (which can be used in combination with the step-wise cooling described above) is that of forming the spheroids by a period of orbital rotation at a first speed followed by a period of orbital rotation at a second speed which is higher than the first speed.
  • the orbital rotation occurs in a generally horizontal plane.
  • the first speed may be at least 50 rpm and the second speed may be at least 1 rpm higher than the first speed (e.g. from 1 to 10 rpm higher, preferably from 1 to 5 rpm higher).
  • the first speed is from 50 to 90 rpm (e.g. from 60 to 80 rpm). Most desirably it is up to 76 rpm, e.g. about 75 rpm.
  • the second speed is from 60 to 90 rpm (e.g. from 65 to 85 rpm). Most desirably it is about 77 rpm.
  • orbital rotation at each of the first and the second speeds may be for a period of at least 24 hours. The total period of orbital rotation prior to cryopreservation would generally be from 2 to 20 days. (Such a period is also suitable if, less preferably, orbital rotation at only a single orbital rotation speed is used.)
  • the spheroids are thawed. This can be done by placing containers containing the spheroids in a water bath at 37 °C, typically for at least 2-3 mins. Once thawed, the cryopreservation medium can be removed e.g. by centrifugation. The spheroids may then be washed and further traces of the cryopreservation medium removed (e.g. by centrifugation).
  • orbital rotation is also performed after thawing of the spheroids. This can be done at one or more rotation speeds of at least 50 rpm (e.g. of from 50 to 90 rpm). If two or more different orbital rotation speeds are used then a period of rotation at a first speed will usually be followed by a period of rotation at a second speed which is higher than the first speed.
  • the spheroids are rotated (post-cryopreservation) at a rotation speed of from 50 to 70 rpm (e.g. of about 60 rpm). Typically this may be done for at least
  • the thawed spheroids can generally be maintained in cultures post-cryopreservation for a period of around 7-10 days (longest period so far tested). Maintenance in a viable state for longer periods than this is however possible.
  • a typical culture medium would include a balanced salt solution and glucose.
  • DMEM:F12 (3:1) plus 10 mg transferrin/100 ml (Sigma), 0.5 mg Insulin/100 ml, 30 nM L-triiodo-thyronine can be used (DMEM is Dulbecco's Minimal Essential Medium and F12 is Hams F12 Nutrient).
  • cryopreservation of avian embryo whole brain spheroids was optimal using the cryoprotectant DMSO (15%), as determined by comparison of fresh spheroids and those recovered after cryopreservation; assessment relied upon a comprehensive range of parameters (morphological, histological and biochemical).
  • the post-thaw rotation speed was shown to have a significant effect on the 'recovery' of spheroid morphology.
  • the presence of antioxidants, e.g. vitamin E appeared to have a beneficial effect on maintaining certain enzyme levels, e.g.
  • the spheroids can be used for any desired purpose.
  • the present invention provides an apparatus adapted to agitate material (e.g. cells, viruses or other biological material) present within a fluid i.e. to move said material within said fluid.
  • the apparatus comprises a self-contained power source (e.g. one or more batteries) and is preferably rechargeable.
  • the apparatus may be adapted to impart any desired form of motion to said material within the fluid (e.g. random motion, motion, side-to-side motion, up-and- down motion, yawing, pitching, rolling, etc.
  • any desired form of motion e.g. random motion, motion, side-to-side motion, up-and- down motion, yawing, pitching, rolling, etc.
  • the apparatus of the present invention is advantageous in that it can be used in a sealed chamber (e.g. an incubator) without the need for connection to an external power supply - i.e. it can be used in stand-alone form. Sealed chambers are often used to reduce or minimise contamination of a sample. They may be provided under sterile or near-sterile conditions.
  • the apparatus may be provided with a container and means may be provided for releasably attaching the container to the apparatus.
  • the container is adapted to hold one or more samples that are to be processed using the apparatus.
  • the samples may be present in culture vessels. These may be well plates, e.g. 96-well plates.
  • the container may be adapted to hold a plurality of such plates in stacked form.
  • the apparatus and container may be provided together in a kit that optionally includes instructions for mounting the container to the apparatus.
  • the kit may also optionally include one or more culture vessels, such as well plates, one or more rechargeable batteries, and an electrical connector.
  • FIGURE 1 shows the glucose consumption (corrected for Protein) before and after cryopreservation in DMSO or in DMSO + Vitamin E for chick Brain spheroids grown in a serum-free medium.
  • FIGURE 2 shows the Protein content of spheroids before and after cryopreservation in DMSO or DMSO + Vitamin E for chick brain spheroids grown in a serum-free medium.
  • FIGURE 3 shows the Acetylcholinesterase (AChE) activity (corrected for protein) before and after cryopreservation in DMSO or in DMSO + Vitamin E for chick brain spheroids grown in a serum-free medium.
  • AChE Acetylcholinesterase
  • FIGURE 4 shows the lactate dehydrogenase (LDH) activity/content (corrected for protein) before and after cryopreservation in DMSO or DMSO + Vitamin E for chick brain spheroids grown in a serum-free medium.
  • LDH lactate dehydrogenase
  • FIGURE 5 shows a comparison of different DMSO concentrations (5-15 % DMSO) on LDH leaking into the culture medium (i.e. released from spheroids) after cryopreservation and at different times after cryopreservation for chick brain spheroids cultured in a serum-free medium.
  • FIGURE 6 shows the glutathione (GSH) level (corrected for protein) before and after cryopreservation in DMSO or DMSO + Vitamin E for chick brain spheroids grown in a serum-free culture medium.
  • GSH glutathione
  • FIGURE 7 shows the glucose consumption (corrected for protein) before and after cryopreservation in DMSO or in DMSO + Methylcellulose for chick brain spheroids grown in a serum-supplemented culture medium.
  • FIGURE 8 shows the protein content of chick brain spheroids grown in a serum-supplemented culture medium following cryopreservation in DMSO or in DMSO + Methylcellulose.
  • FIGURE 9 shows the Acetylcholinesterase (AChE) activity (corrected for protein) before and after cryopreservation in DMSO or in DMSO + Methylcellulose for chick brain spheroids grown in serum-supplemented culture medium.
  • FIGURE 10 shows the lactate dehydrogenase (LDH) activity/content (corrected for protein) before and after cryopreservation in DMSO or in DMSO + Methylcellulose for chick brain spheroids grown in a serum- supplemented culture medium.
  • AChE Acetylcholinesterase
  • FIGURE 11 shows the glutathione (GSH) content (corrected for protein) before and after cryopreservation in DMSO or in DMSO + Methylcellulose for chick brain spheroids grown in a serum-supplemented culture medium.
  • GSH glutathione
  • FIGURE 12 gives a schematic representation of the development and use of cryopreserved brain and liver spheroids for use in toxicity screening and testing and/or neuroefficacy testing of new compounds.
  • Cells for preparing the spheroids could be derived from a number of species and cocultured with other cell types (e.g. endothelial cells for the blood-brain barrier).
  • spheroids from liver and brain prepared independently could be cocultured to investigate metabolic activation of compound.
  • the spheroids, or cells used to reaggregate to spheroids could be also transfected with genomic reporter-promoter constructs for toxicity/efficacy biomarkers to facilitate the screening process and event marking
  • the cells Following cryopreservation (independently, or together as shown in figure 12) the cells would be exposed to test compounds and then assessed either using the reporter gene markers, or other assays including cytotoxicological, morphological or neurotoxicologial/neurochemical markers.
  • FIGURE 13 outlines a proposed methodology for identifying relevant molecular toxicity biomarkers and incorporating reporter gene constructs for these markers into spheroids.
  • Spheroids would be exposed to the test agent, mRNA isolated and a differential mRNA display produced by reverse transcripase PCR to enable identification and cloning of the response gene(s). The promoter element would then be identified and isolated.
  • the dissociated cells from the desired tissue could be transfected with a reporter-promoter construct using a variety of technologies (e.g. electroporation, adenoviral vectors, etc.) and the spheroids prepared incorporating these constructs.
  • the response would be detected optimally by designing a 96 well spheroid culture format (e.g. 1 spheroid/ well).
  • FIGURE 14 The plates in this figure show spheroids from chick brain before (at 7 DIV) and after cryopreservation following 24 hours in re- culture as described in experiment 1, using 15% DMSO as the cryopreservant. The images were similar in the presence of Vitamin E or Methylcellulose additives with the DMSO.
  • FIGURE 15 The plates in this figure show spheroids from chick brain before (at 7 DIV) and after cryopreservation following 7 days reculture as described in Experiment 1, using 15 % DMSO as the cryopreservant. These images were similar in the presence of Vitamin E or Methylcellulose additives with the DMSO.
  • FIGURE 16 shows in schematic form a liver and brain spheroid co-culture.
  • FIGURE 17 shows a rechargeable apparams that can be used in the preparation of spheroids.
  • Examples A to E describe the general methodology which can be used to prepare and analyze fresh chick and rat brain spheroids.
  • a method for the production of liver spheroids and the apparatus for liver-brain spheroid co-culture is discussed in Example F below.
  • Example G describes a rechargeable apparams that can be used in the preparation of spheroids.
  • Pregnant female Wistar rats are sacrificed at 15-16 days of gestation (Atterwill, 1987).
  • the uteri are removed to a sterile laminar flow cabinet and the dissection carried out under aseptic technique.
  • the foetuses are removed from the uteri and whole brains including diencephalon and midbrain are removed by a single cut across the top of the head and the brain scooped out into ice-cold isotonic Hanks
  • the tissue is gently extruded through the filter.
  • the suspension is then filtered by gravity through a 118 mm filter, producing a single cell suspension and removing contaminating meninges.
  • the resultant suspension is centrifuged at 150 g for 5 min.
  • the supernatant is discarded and the cells triturated with 10 ml fresh Dl .
  • the cells are then centrifuged and triturated once more. After the third centrifugation the cells are triturated in 10 ml growth medium (Dulbecco's modified eagles medium(DMEM) supplemented with 10% fetal calf serum, 5 ml L-glutamine and 25 mg/ml gentamycin).
  • DMEM Dulbecco's modified eagles medium
  • the cells are counted and diluted to give a density of 1 x 10 7 cells/ml.
  • 3.5 x 10 7 cells are added to 25 ml capacity Delong flasks.
  • Spheroid cultures are grown in a 9% CO 2 /humidified air incubator at 37°C on a gyrotatory shaker (New Brunswick) at 85 rpm.
  • DIV in vitro
  • small spheroids After two days in vitro (DIV) small spheroids have formed and these are transferred to 50 ml Delong flasks and 5 ml fresh growth medium added. Medium is changed on alternate days by removing half the old and replacing it with fresh medium.
  • DIV spheroids are transferred into 6-well plates (Nunc) in a final volume of 2 ml, except for those spheroids which are to be assayed for lipid peroxidation. Treatments are performed on 12 DIV and repeated on 14 DIV at 50
  • Spheroids are harvested and homogenised at 15 DIV for the TBA assay and at 16 DIV for all other assays.
  • growth media (1 ml) is removed from each well for LDH analysis. This is centrifuged at 13000g prior to freezing at -20 °C.
  • the cultures are then transferred to 1.8 ml microcentrifuge tubes and washed three times in PBS at 37, 20 and 4°C respectively to avoid heat shock.
  • the cultures are kept on ice prior to homogenising in homogenising buffer (NaH 2 PO 4 , 2 mM; EDTA, 0.5 mM; NaCl, 145 mM).
  • the suspension is then aliquoted ready for analysis. The aliquots are stored at -70°C.
  • Chick brain spheroid cultures are prepared using a procedure modified significantly from that first described by Wylegyurech and Reinhardt (1991). Fertile eggs from White-leghorn hydrid hens are incubated for seven days at 37° C in a humidified egg incubator (Curfew). Embryos at approximately stages 27-29 (Hamburger and Hamilton, 1951) are removed into isolation buffer consisting of NaCl, 128.5 mM; KC1, 5.4 mM; glucose, 5.5 mM; sucrose, 51.8 mM; HEPES, 25 mM; BSA 0.1 %; pH 7.4 (Bruinink et al, 1987) at room temperamre and washed three times in isolation buffer.
  • Cells are diluted in culture medium (various, see later) to give a final concentration of 1.33 x 10 6 viable cells/ml and plated out into 6 well pates (Nunc) with 3 ml per well. Cultures are incubated at 37 °C in a humidified incubator with 5 % CO 2 on a gyrotatory shaker (New Brunswick) at 80 rpm. Currently cell viability varies between 70-80% with each preparation, with approximately 1.5-2.5 x 10 7 cells per chick brain. Typically, each culture well contains between 800-1100 spheroids by 14 DIV, and one well yields 1-1.4 mg of total protein. Spheroid development is monitored by measurement of diameter using a graticule under light microscopy.
  • DMEM fetal calf serum
  • DMEM/HAMS F12 a serum-free formulation of DMEM/HAMS F12 (3: 1) supplemented with Bottensteins' and Sato's N3 neural cells supplement, to give a final concentration of the following: transferrin, human holo-form 50 mg/1; insulin, bovine 5 mg/1; progesterone, 20 nM; selenium, 30 nM, and putrescine, 100 ⁇ M (Bottenstein, 1985). Serum-free media is also supplemented with 30 ⁇ M L-triiodo-thryronine (LT3) after Atterwill et al (1984).
  • LT3 L-triiodo-thryronine
  • Chick brain spheroid culmres are harvested as described for whole rat brain spheroids and similar assays conducted on spheroid homogenates (see later). Expression of AChE, GFAP, GS, LDH, glucose consumption and total protein are followed over a period of culmre up to 30 DIV in several media formulations. Spheroids are harvested at various intervals after inoculation, washed in PBS (pH 7.4) and samples homogenised and frozen at -70°C. Monolayer culmres are used for initial 'dose-ranging' cytotoxicity measurements, and spheroids exposed to non-cytotoxic concentrations in order to assess neurotoxicity. Spheroid homogenates/supernatants are assayed for measures of cytotoxicity and neurotoxicity (neurone- and glial-specific markers) and spheroids processed for histology (paraffin and frozen sections).
  • markers of specific and general toxicity are required. This can be achieved using both biochemical assays (Honegger and Richelson, 1976) and histological techniques (Trapp et al, 1979). Biochemical markers for both neuronal and glial cell function have been measured. Acetylcholinesterase (AChE), which is found in cholinergic neurones has been measured to estimate neuronal toxicity (Honegger and Richelson, 1976) and it is also used as a specific marker for organophosphate toxicity (Vargese et al, 1995).
  • AChE Acetylcholinesterase
  • Astrocyte function is assessed by the measurement of the glial specific enzyme, glutamine synthetase (GS) (Martinez-Hernandez et al, 1977) and glial fibrillary acidic protein (GFAP) which is a marker of reactive gliosis (Eng, 1985).
  • Oxidative stress is determined by measurement of lipid peroxidation (Aust and Buege, 1978). Release of lactate dehydrogenase (LDH), a cytosolic enzyme into the media is utilised as a marker of general cytotoxicity. All results are generally normalised against total protein.
  • Protein Assay Total protein is assessed according to the method of Bradford (1976).
  • Homogenates are diluted, 1:2, and 10 ⁇ l added per well of a microtitre plate.
  • the Coomassie blue protein reagent (Bio-Rad) is diluted 1:5 with Millipore water and filtered through Whatman No. 1 filter paper.
  • the diluted reagent is added to the homogenate (200 ⁇ lper well) and mixed well.
  • the plate is allowed to stand for 5 min at room temperamre prior to recording the absorbance at 595 nm.
  • Acetylcholinesterase Assay (AChE)
  • AChE activity is measured according to the method of Ellman et al (1961). 100 ⁇ l of sample is placed in each well of a 96- well plate with 100 ⁇ l sodium phosphate buffer (0.1 M, pH 7.4,), 50 ⁇ l 5,5'-dithiobis-2-nitrobenzoate (DTNB)
  • acetylthiocholine iodide (2 mg/ml in methanol,) and 25 ⁇ l acetylthiocholine iodide (2.5 mg/ml in buffer,).
  • the resultant absorbance is monitored at 405 nm for 15 min, at 10 sec intervals, on a Biotek EL312 kinetic plate reader.
  • Activity is calculated using a standard curve of pure acetylcholinesterase (AChE) from electric eel (Sigma).
  • AChE can hydrolyse 1 mmole of acetylcholine to choline and acetate per minute at pH 8.0 and 37°C.
  • GS is assayed using the transf erase site of the enzyme, as reported by Thorndyke and Rief-Lehrer (1976).
  • Homogenate 80 ⁇ l is transferred to a microcentrifuge tube and 330 ⁇ l reagent mix containing 0.4 M L-glutamine; 1.0 M hydroxylamine; 395.0 mM potassium arsenate; 823.0 mM sodium citrate; 82.3 mM manganese chloride; 4.0 mM adenosine diphosphate was added.
  • the tubes are mixed and then incubated in a shaking water bath at 37°C for 15 mins.
  • the reaction is terminated on ice by the addition of 400 ⁇ l FeCl 3 solution (1: 1: 1 mix of 7% FeCl 3 in 5 mM HC1: 15% TCA: 0.1 M HC1) .
  • the mixtures are then centrifuged at 13000 g and absorbance at 500 nm measured. Results are compared against a standard of purified GS from sheep brain.
  • One unit of GS is defined as that which will convert 1.0 mmole of
  • GFAP Glial Fibrillary Acidic Protein Assay
  • GFAP is estimated using an adaptation of O'Callaghan's (1991) method. Immulon-2 plates are coated overnight at 4°C with mouse monoclonal GFAP antibody (1:1000). Excess antibody is removed by washing with PBS. All subsequent incubations are carried out at room temperamre on a rotatory shaker. Non-specific binding to the plastic is prevented by incubation with 5 % milk powder in PBS. This is washed off with PBS, and a 100 ⁇ l sample or standard added and left to incubate for two hours. Samples are centrifuged (13000 g) and diluted 1:2 before use. The plates are then washed with PBS-TWEEN (PBS and 0.05% TWEEN-20).
  • Rabbit-derived GFAP antiserum diluted 1:500 in BLOTTO (5% milk powder in PBS-TWEEN) is then added and left to incubate for 1 hr. After washing with PBS-TWEEN, anti-rabbit immunoglobulins conjugated to alkaline phosphatase are added, diluted 1:3000 in BLOTTO, for 1 hr. The wash step is repeated and finally 1 mg/ml p-nitrophenyl phosphate in 10% diethanolaminebuffer, the substrate for alkaline phosphatase, is added. This substrate is converted to p-nitrophenol, a yellow adduct which has an absorbance maxim at 405 nm, the absorbance being proportional to the concentration of GFAP in the sample.
  • BLOTTO 5% milk powder in PBS-TWEEN
  • anti-rabbit immunoglobulins conjugated to alkaline phosphatase are added, diluted 1:3000 in BLOTTO, for 1 hr.
  • the wash step is repeated
  • Lipid peroxidation is assessed using the thiobarbituric acid (TBA) assay (Aust and
  • the homogenates are then centrifuged at 150 g for 5 mins to remove any proteins and the absorbance of the resultant pink chromophore monitored at 532 nm.
  • a standard curve of mM concentrations MDA (malondialdehyde bis (diethyl acetal)) against resultant absorbance is used to standardise results.
  • LDH LDH released from spheroids compared to total cellular LDH according to the method of Korzeniewski and Callewaert (1983). Samples (100 ⁇ l per well) are added to 96-well plates along with 100 ⁇ l "LDH buffer” (0.2 M Tris buffer, pH 8.2; L(+) lactate, 54 mM; ⁇ NAD, 1.3 mM; phenazine methosulphate,
  • NTE Neuropathy Target Esterase
  • the assay for NTE in chick brain spheroids is based on the methods of Johnson
  • Phenol release by hydrolysis is complexed with 4-aminoantipyrine to give an orange reaction product which is read spectrophotmetrically at wavelength 500 nm.
  • Chick brain spheroids are harvested as described previously, washed three times in phosphate buffered saline (PBS, pH 7.4) and resuspended in 0.5 ml of Tris/EDTA buffer (Tris, 50 mM; EDTA, 0.2 mM; pH 8). Spheroids are then homogenised in a motorised Potter homogeniser (10 strokes at 800 rpm), and total protein measured using the Biorad Coomasie blue assay with a gamma-globulin standard (Bradford, 1976), and then diluted to give a final concentration of 0.3 mg/ml protein in Tris/EDTA.
  • PBS phosphate buffered saline
  • Tris/EDTA buffer Tris, 50 mM
  • EDTA 0.2 mM
  • pH 8 Tris/EDTA buffer
  • Spheroids are then homogenised in a motorised Potter homogeniser (10 strokes at 800 rpm), and total protein measured using the Biorad Coomasie blue
  • Triplicate 50 ⁇ l aliquots of homogenate are plated into 96 well plates (Nunc) and incubated with either paraoxon (40 ⁇ M), paraoxon (40 ⁇ M) with mipafox (50 ⁇ M), or buffer (tissue blank). Plates are mixed using a Labsystems Multiscan RC plate reader for 30 sec and incubated for 20 min at 37 °C. Following incubation, 50 ⁇ l of a 1.06 mg/ml suspension of phenyl valerate in 0.03 % Triton X100 in water is added to wells containing mipafox and/or paraoxon and 50 ⁇ l of Triton XI 00 to tissue blanks.
  • NTE activity in the sample homogenates of chick brain spheroids is expressed as ⁇ moles of phenol released per minute per mg of protein, and is determined by subtracting the phenol concentration of tests containing mipafox and paraoxon from those containing paraoxon alone.
  • Glucose in culture media is the major energy source for the spheroids and glucose consumption generally reflects the metabolic status and viability of the culmres.
  • Glucose is measured in spheroid culture supernatant using a glucose kit (Sigma) based on an enzymic assay. The method is modified in our laboratories for use in a 96-well plate format. A 100 ⁇ l aliquot of media is taken at the end of a defined period of incubation (usually 24 hours) and diluted 1 :50 in distilled water. A 50 ⁇ l sample of diluted culture media or glucose standards are added to a 96 well plate and 250 ⁇ l of colour reagent added to each well and incubated at 37 °C for 30 min.
  • Plates are read spectrophotometrically at wavelength 450 nm.
  • chick brain spheroid culmres single cells grow together to form three-dimensional spheroids and during this reorganization some cells locate in the inner part of the spheroid while others form the outer surface.
  • the geographical isolation of the inner cells from the culture medium means that there will be a gradient of oxygen consumption across the spheroid (Breedel-Geissler et al, 1992), which would suggest that energy metabolism in the inner part is lower than that in the outer cells. Variations on this procedure for cryopreserved spheroid assessment is given in the next section.
  • Spheroids are fixed in 2 ml formal saline (10% formalin in 0.9% NaCl) for 30 min. The spheroids are then dehydrated by exposure to increasing concentrations of ethanol (70%, 90% and absolute). The absolute ethanol incubation is repeated to remove all traces of water and is then replaced with xylene, which enables wax to be added without emulsions forming.
  • the paraffin wax is kept molten at 60°C.
  • the spheroids are washed in wax three times, then fresh wax is added, and transferred to a mould and allowed to set overnight. Sections of 7 ⁇ m are cut and fixed onto subbed slides.
  • Spheroids are harvested and transferred to 1.5 ml Eppendorf tubes, washed twice in PBS and resuspended in 1 ml phosphate buffered formalin (PBF) and left to fix at room temperamre overnight. Fixed spheroids are washed in PBS and resuspended in 0.5-1 ml molten agarose (1%). When the agarose has solidified, the plugs are removed from the tubes and cut into transverse sections, approximately 5 mm wide. These are fixed overnight in PBF and then processed in a Shandon tissue processor, dehydrated, cleared in xylene and embedded in paraffin wax.
  • PBF phosphate buffered formalin
  • Sections are cut at 9 and 5 ⁇ m using a Shandon rotary microtome and attached to gelatin- or poly-D-lysine-coated slides. Sections are stained with either haematoxylin and eosin or immunohistochemically stained as described for rat brain spheroids.
  • Haematoxylin and Eosin Staining Slides are rehydrated by incubating with xylene, absolute alcohol, 90% and 70% alcohol, followed by PBS.
  • Ehrlichs haematoxylin which stains negatively charged chromatin within the nuclei, is added to each slide and left for 10 min. Excess haematoxylin is washed away with distilled water until the section turns blue. The slide is then exposed to the counterstain eosin, for 5 min. Excess stain is removed with 70% alcohol, followed by absolute alcohol and xylene prior to mounting in
  • the slides are incubated for 30 min with a 1:400 dilution of biotinylated anti-mouse immunoglobulin.
  • the slides are then incubated with a solution of avidin and biotinylated horseradish peroxidase for 30 min.
  • the slides are again washed, and the substrate, 3,3' diaminobenzidine tetrachloride/urea added to form a brown precipitate.
  • the slides are then dehydrated with 70%, 90%, absolute ethanol and xylene and embedded in DPX.
  • liver spheroids from rat liver material
  • co-culture technology for brain and liver spheroids. Modifications of this technology can be used for liver spheroids from other species prior to cryopreservations.
  • a standard 'hepatocyte isolation rig' is necessary. This basically consists of three flasks connected, via a peristaltic pump, to a platform with two cannulae.
  • the flasks and tubing Prior to liver collection, the flasks and tubing are rinsed with 70% IMS followed by sterile 'perfusion buffer'. The water bath is also switched on.
  • the rig is loaded with 500 ml 'chelating buffer' in flask 1, 250 ml 'perfusion buffer' in flask 2 and 100 ml 'collagenase buffer' in flask 3 (see appendix). All tubes are primed and the whole system finally primed with 'chelating buffer' .
  • a Male rat is killed by cervical dislocation. The abdomen is washed, and a long horizontal incision is made just below the rib cage. The liver is cut free from the connective tissue whilst being held over a beaker containing 'perfusion buffer' (see appendix).
  • the two largest lobes of the liver are taken and the cannulae inserted into the open, cut blood vessels.
  • 'Chelating buffer' is allowed to flow (5 ml/min/cannula) and an almost immediate clearing of blood from the lobe should be seen. This is allowed to run for 15 mins.
  • the tap is changed to allow the 'perfusion buffer' to flow, again for a further 15 mins. Nearing the end of this period the collagenase (see appendix) is dissolved in a little 'collagenase buffer' and returned to flask 3.
  • the 'collagenase buffer' is allowed to perfuse for 30 mins, with recycling of the buffer by placing the waste mbe in flask 3. After this time the liver should appear spongy and digested, if not leave it for longer.
  • the lobes are removed into sterile petri dishes.
  • Dulbecco's Modified Essential Media (see appendix) is added to the lobes in separate dishes.
  • the liver is teased apart with forceps and a spatula and the cells should flow into the media. Do not dissociate any badly digested parts of the liver.
  • the cell suspension is poured through a 63 ⁇ m Nybolt filter into a centrifuge mbe and the petri dish washed. The cell suspension is centrifuged at
  • Hepatocyte viability is assessed by trypan blue exclusion. A 1:5 dilution of the hepatocyte suspension is usually necessary, followed by the addition of 200 ⁇ l trypan blue (0.4%) to 200 ⁇ l cell suspension.
  • Viable cells remain clear whilst dead cells stain blue. A viability of 80-85% is expected, below 70% should be discarded. There is usually a difference between the two lobes and the best viability suspension is used.
  • Cell count (cells/ml) no. cells counted x dilution factor (10)x 10 4 .
  • Perfusion buffer 435 ml sterile 'millipore water' 50 ml Earles balanced salt solution xlO (without Ca + ,
  • Chelating buffer 490 ml perfusion buffer
  • Collagenase buffer 100 ml perfusion buffer
  • the plates need to be coated with poly(-2-hydroxyethylmethylacrylate) (p-HEMA) (Sigma) to prevent the cells adhering to the plastic.
  • p-HEMA poly(-2-hydroxyethylmethylacrylate)
  • a 2.5% w/v solution of p-HEMA is made in 95% absolute ethanol. This needs to be left to stir on a gently heated hot plate (50 °C) and will take around 3 hrs to dissolve.
  • 6-well tissue culmre plates are coated by adding 2ml p-HEMA solution to each well and allowing them to air dry in a class II safety cabinet. Once dry (approx. 3hrs) these can be stored at 4°C.
  • Culmre procedure An isolated hepatocyte cell suspension is obtained by a collagenase perfusion method. Cells are plated at a density of between 2.5-5.0 x 10 5 cells/ml (still under investigation), at 2 ml suspension per well of a 6-well p-HEMA coated plate.
  • Culmres are maintained in an incubator at 37 °C, 9% CO 2 and are gently rotated at between 85-90 rpm.
  • Culture media 425 ml sterile millipore water
  • the culmres are sampled for viability and functional assays. Two random wells are combined to give one sample. 500 ⁇ l of media is removed from each well and combined, microfuged and the supernatant frozen at -80°C for LDH and albumin assay. The spheroids are transferred from the well into a 1.5 ml eppendorf. The well is washed and the washings transferred. The spheroids are allowed to settle and the supernatant removed (checking for tissue loss under the microscope). The contents of one well are resuspended in 1 ml 'homogenising buffer' and this is transferred to the contents of the second well.
  • the spheroids are allowed to settle again, the supernatant removed and finally resuspended in 1 ml homogenising buffer.
  • the culture samples are sonicated, to disrupt the cells, with a probe for 10 sees and stored on ice. 200 ⁇ l aliquots of the samples are stored at -80°C until assay.
  • Homogenising buffer - see rat brain spheroid culmre method.
  • a rechargeable apparams that can be used in the preparation of spheroids is within the scope of the present invention. It is shown in Figure 17 and is discussed below.
  • Figure 17 shows an apparams (1) that is adapted to allow orbital rotation of cells suspended in a fluid. Desirably this rotation occurs in a generally horizontal plane. However the apparams (1) may also be adapted to allow orbital rotation in other planes and may even be adapted to allow other forms of movement (e.g. side-to-side shaking, rolling, pitching, yawing, etc). This can be advantageous if the apparams (1) is to be used for other purposes than for forming spheroids.
  • the apparams (1) may also be adapted to allow orbital rotation in other planes and may even be adapted to allow other forms of movement (e.g. side-to-side shaking, rolling, pitching, yawing, etc). This can be advantageous if the apparams (1) is to be used for other purposes than for forming spheroids.
  • the apparams (1) is provided with a control panel (6). This allows the rate of orbital rotation of cells to be maintained within a desired r.p.m. range (preferably 50-90 r.p.m.).
  • An indicator (3) is also provided which allows r.p.m. values to be displayed.
  • a removable plate (5) is shown, behind which a rechargeable power source (e.g. one or more rechargeable batteries) is located.
  • a port (4) is provided to allow a detachable electric cable to be attached to the apparatus (1) so that the power source can be recharged.
  • the cable can be removed.
  • the apparams (1) can therefore be used in stand-alone form (i.e. without the need for it to be connected to an external power source). This is advantageous in that the apparams (1) can be used in a sealed chamber that need not itself be provided with a power source. Such chambers are often used in order to minimise contamination of a sample. They may be maintained under sterile or near-sterile conditions.
  • a container (2) may be mounted to the apparams (1) via releasable securing means (not shown).
  • the container (2) adapted to contain one or more culmre plates.
  • a single 96 well culmre plate (8) is shown, but a plurality of such plates may be stacked in the container (2).
  • the container (2) is mounted onto a platform of the apparams (2).
  • the platform is operatively connected to a motor (not shown) to allow the platform to be moved in a desired manner. Controlled movement of the platform is used to impart movement to cells in suspension so that spheroids can be formed. Thus orbital rotation of cells can be imparted via orbital rotation of the platform.
  • the apparams includes a plurality of legs (7), which may be provided in a form so that their heights can be adjusted. This is advantageous if it is desired for the platform (not shown) to be generally horizontal since this can be achieved by adjusting the legs (7) accordingly.
  • cryopreserved chick brain spheroids can be prepared taking as a starting point the general methodology described in the following examples.
  • the cells were counted and diluted with incubation medium to give a density of 1.33 x 106 cell/ml; 3ml diluted suspension was added to each well of 6-well plates.
  • the incubation medium contains 300 ml Dulbeccos Modified Eagle's Medium (DMEM) x 1, 100 ml F12 (Ham), 20 mg transferrin, 2 mg insulin, 1.6 nmol progesterone, 40 ⁇ mol putrescine, 1.2 nmol selenium, 48 ⁇ g triiodothyronine and 5 ml penicillin and streptomycin.
  • DMEM Dulbeccos Modified Eagle's Medium
  • the vials are taken out from liquid nitrogen and thawed at 37 °C in a water bath for 2-3 min. They are centrifuged at 800 rpm for 1 min and the cryopreservative medium removed.
  • Spheroids are washed once with 1 ml incubation medium and last step repeated. Then transferred to 6-well plate with 3 ml medium/vial/well. The plates are shaken at 60 rpm in an incubator at the standard incubation condition. The incubation is terminated at either 24 hours or 3 or 7 days after recovery from freezing for biochemical assays.
  • Glucose assay is carried out by using Glucose Kit (Sigma, Catalog No. 510- A) which is based on Keston's enzymatic method (Keston, 1956). The method was modified in our laboratory to be suitable for 96-well Plate Reader assay which is quicker and more economic than the conventional method. Medium of 100 ⁇ l/well is taken at the end of a defined period of incubation (usually 24 hours) and diluted with distilled water (1:50). A blank control well must be used as the reference value for consumption calculation.
  • Spheroids were homogenated in phosphate buffer (pH 7.4). Total protein was assayed according to the method of Bradford (1976). 10 ⁇ l homogenates were added per well of 96-well plate. The protein reagent (Bio-Rad) was diluted 1:5 with millipore water and filtered through Whatman No, 1 filter paper. The diluted reagent (200 ⁇ l per well) was added to the homogenate and mixed well. The plate was allowed to stand for 5 min at room temperamre prior to recording the absorbency at 595 nm.
  • AChE activity was measured according to the method of Ellman et al (1961). 100 ⁇ l of homogenate was placed in each well of a 96-well plate with 100 ⁇ l sodium phosphate buffer (0.1 M, pH7.4), 50 ⁇ l 5,5' -dithiobis-2-nitrobenzoate (DTNB) (2 mg/ml in methanol) and 25 ⁇ l acetylthiocholine iodide (2.5 mg/ml in sodium phosphate buffer). The resultant absorbency was monitored at 405 nm for 15 min, at 10 sec. intervals, on a Biotek EL312 kinetic plate reader.
  • DTNB 5,5' -dithiobis-2-nitrobenzoate
  • Activity was calculated using a standard curve of pure acetylcholinesterase from electric eel (Sigma).
  • One unit of AChE can hydrolyse 1 ⁇ mole of acetylcholine to choline and acetate per minute at pH 8.0 and 37 °C.
  • ⁇ l homogenate was diluted with equal volume of 13 % trichloroacetic acid (TCA). Vortex mixing and then centrifuged at 13,000 rpm for 3 min. 75 ⁇ l supernatant or standard was added to 2775 ⁇ l of phosphate/EDTA buffer (1 litre containing 13.6 g KH2PO4, 1.86 g EDTA, pH is adjusted to 8 with NaOH) and 150 ⁇ l of 0.1 % o-phthaldehyde (OPT) in methanol was added. The samples were mixed and kept at room temperamre for 30 min. They were read on a fluorimeter at 350 nm exitation and 420 nm emission.
  • TCA trichloroacetic acid
  • the cells were counted and diluted with incubation medium to give a density of 1.33 x 10 6 cell/ml.
  • the incubation medium contains 10% foetal calf serum in the mixture of Dulbeccos Modified Eagle's Medium (DMEM) x 1 and F12 (Ham) (3: 1) and 1 ml penicillin and streptomycin per 100 ml medium. 3 ml diluted suspension was added to each well of 6-well plates. The plates were then placed on a shaker in 37°C 5 % CO 2 incubator. The rotation speed of the shaker is adjusted to 75 rpm for the first 4 days and then to 77 rpm thereafter. III) Cryopreservation
  • the vials are taken out from liquid nitrogen and thawed at 37 °C in a water bath for 2-3 min. Then they are centrifuged at 800 rpm for 1 min and the cryopreservative medium removed. Spheroids are washed once with 1 ml incubation medium and the last step repeated. Then spheroids are transferred to 6-well plate with 3 ml medium/vial/well. The plates are shaken at 60 rpm in an incubator for 24 hour and then adjusted to 75 rpm until the termination of the experiment. The incubation was terminated at either 24 hours or 3 or 7 days after recovery from freezing for biochemical assays.
  • Figs 1-6 and Table 1 The results depicted in Figs 1-6 (and Table 1) show the viability of chick brain spheroids grown initially in a serum-free based medium at -43- various timepoints after cryopreservation (as described in Methods) using either DMSO alone, or DMSO + Vitamin E. All parameters were corrected for protein content.
  • Fig. 2 shows that the cryopreservation procedure reduced protein content to about 20% of original values indicate loss of a large number of the spheroids by the cryopreservation process. This was backed by a reduced lactate dehydrogenase (a soluble cytoplasmic marker) (LDH) spheroid content (Fig 4.). However, as Fig.
  • the orbital shaking speed has a significant influence on both spheroid growth or recovery from the cryopreservation state.
  • the orbital shaking speed at 75 rpm for first 4 days and then 77 rpm before freezing achieved a better formation of spheroids. 60 rpm after recovery significantly reduced broken spheroids.
  • the cells were counted and diluted with incubation medium to give a density of 1.33 x 10 6 cell/ml.
  • the incubation medium contained 10% foetal calf serum in the mixmre of Dulbeccos Modified Eagle's Medium (DMEM) x 1 and F12 (Ham) (3:1) and 1 ml penicillin and streptomycin per 100 ml medium. 3 ml diluted suspension was added to each well of 6-well plates. The plates were then placed on a shaker in 37 °C 5% CO 2 incubator. The rotation speed of the shaker is adjusted to 75 rpm for 4 days and then to 77 rpm for 7 days.
  • DMEM Dulbeccos Modified Eagle's Medium
  • Ham Ham
  • the vials were centrifuged at 800 rpm for 1 min. The medium was carefully removed and then 1 ml cryopreservative medium added containing 15% DMSO and 0.1% methylcellulose (SIGMA) in 10% CFS medium at 4°C. The vials were kept at this temperamre for 40-60 min and then transported to a -20 °C freezer. After 2 hours freezing at -20° C, the vials were plunged into liquid nitrogen (-196°C).
  • SIGMA methylcellulose
  • the vials were taken out from liquid nitrogen and thawed at 37° C in a water bath for 2-3 min. Then they were centrifuged at 800 rpm for 1 min and the cryopreservative medium removed. They were washed once with 1 ml incubation medium and the last step repeated. They were then transferred to a 6-well plate with 3 ml medium/vial/ well. The plates were shaken at 60 rpm in incubator for 24 hours and then adjusted to 75 rpm until termination of the experiment. The incubation was terminated at either
  • Figs 7-11 and Table 2 The results depicted in Figs 7-11 describe the viability of chick brain spheroids at various timepoints after cryopreservation following pre- cryopreservation culmre in the serum-based culmre medium and using either DMSO alone (15%) or DMSO + Methylcellulose (0.1%) as the cryopreservants. Furthermore, in this experiment the spheroids were rotated at 60 rpm for the initial 24 h following storage and then 75 rpm until experiment termination. All parameters were corrected for protein values.
  • the glucose consumption parameter was approximately halved by the cryopreservation procedure and slightly improved by DMSO and methylcellulose at the 24 h post- cryopreservation timepoint.
  • the absolute pre-cryopreservation value for this parameter was higher in this experiment than in Experiment 1 (presumably due to the improved maturity of spheroids due to serum addition)
  • the reduction in viability as judged by this parameter was lower by the cryopreservation procedure possibly due to a more resricted access of cryopreservant into the spheroids due to their enhanced size in the serum-based culmre enviroment.
  • Fig. 11 shows that glutathione (total GSH) content was not adversely markedly affected by the cryopreservation procedure at 24h and indicates a satisfactory cellular endogenous oxidant protectant level i.e. low oxidant stress level in the spheroids.
  • glutathione (total GSH) content was not adversely markedly affected by the cryopreservation procedure at 24h and indicates a satisfactory cellular endogenous oxidant protectant level i.e. low oxidant stress level in the spheroids.
  • Protein content indicates that around 50% of the spheroids 'seeded' in the initial culmre were lost due to the cryopreservation procedure but of that 50% surviving, the other parameters indicate 50-75% viability overall by this culmre procedure.
  • the acetylcholinesterase activity (Fig. 9) was maintained at around 50% the pre- value showing that, in terms of a specific neuronal (as opposed to glial) marker, neuronal viability is also still fairly good.
  • the LDH activity, however, (i.e. cellular soluble enzyme content) of the cryopreserved spheroids was reduced to approximately 20% of original values in
  • Chick brain spheroids grew faster and have a greater size/diameter in serum-based medium than in serum-free medium.
  • spheroid is taken to mean a three-dimensional strucmre which does not occur in namre and which consists of a reaggregate of cells of a tissue or of an organ.
  • a spheroid will preferably be generally spherical in shape. Desirably it will consist of at least 10 3 -10 4 cells, more desirably of at least 10 3 cells but this may vary according to species and rotation speed and tissue 'type' . Typically a spheroid will have a diameter of between 100 microns and 1000 microns. However it may be larger/ smaller than this. It may be made up of one or more different cell types. If a plurality of different cell types are present they may form different layers of the spheroid.
  • cryopreserving is taken to mean taking the spheroid(s) or cellular aggregate/re-aggregate strucmre down to a cryogenic temperamre (typically -196°C) where the biological sample is held in a state of 'suspended animation' in the presence of a cryopreservant (cryoprotectant), or several cryopreservant (cryoprotectant) chemicals in order to hold the material at this temperamre for prolonged periods (many months, and up to several years) maintaining significant cellular integrity and functionality during the cryopreservation process and upon subsequent retrieval after thawing and return to ambient and physiological temperatures (typically 37°C).
  • a cryogenic temperamre typically -196°C
  • tissue is taken to mean an organised selection of cells having a common function.
  • organ is taken to mean an organised collection of "tissues" having a common function.
  • tissue or organ need not be completely intact to be used in the present invention since parts of whole tissues or organs (which may be obtained via biopsies) can be disrupted to individual cells/small groups of cells before being re-aggregated to form spheroids and then cryopreserved.
  • Atterwill C K Pillar A M, Price A R (1986) The effects of ethylcholine mustard aziridinium (ECMA) in rat brain reaggregate culmres. Br. J. Pharmac. , 88, 355Pa.
  • Atterwill C K (1989) Brain reaggregate culmres in neurotoxicological investigations. In: In vitro Methods in Toxicology. Ed C K Atterwill and C E
  • Atterwill C K and Meakin (1990) Delayed treatment with nerve growth factor (NGF) reverses ECMA-induced cholinergic lesions in rat brain reaggregate culmres. Biochem. Pharm., 39, 2073-2076.
  • NGF nerve growth factor
  • Atterwill C K Davies W J and Kyriakides M A (1990) An investigation of aluminium neurotoxicity using some in vitro systems ATLA, 18, 181-190.
  • Bottenstein J E (1985). Growth and differentiation of neural cells in defined media. In: Bottenstein J E, and Sato G, (Eds) Cell culmre in the neurosciences.
  • Flint O P (1983). A micromass culmre method for rat embryonic neural cells. Journal of Cellular Science, 61: 247-262. Flint O P, and Orton T C, (1984). An in vitro assay for teratogens with culmres of rat embryo midbrain and limb bud cells. Toxicology and Applied Pharmacology, 76: 383-395.
  • Garber B B, and Moscona A A (1972). Reconstruction of brain tissue from cell suspensions I. Aggregation patterns of cell dissociated from different brain regions of the developing brain. Dev Biol, 27, 217-234. George E, Hamilton G, & Westmoreland C, (1996). The use of in vitro models in hepatotoxicity testing. TEN 3 (5).
  • Honegger P Richelson E, (1976). Biochemical differentiation of mechanically dissociated mammalian brain in aggregating cell culmre. Brain Res, 109; 335-354.
  • NTS Neurotoxic esterase
  • Kearney J N Cryopreservation of cultured skin cells. Burns 1991: 17: 380-383.
  • Co-culture of rat embryos and hepatocytes in vitro detection of a proteratogen.
  • Organophosphorus-induced delayed neurotoxicity - a comparative study of the effects of tri-orthyo-tolyl phosphate and triphenyl phosphite on the central nervous system of the Japanese quail. Neurotoxicology, 16: 45-54.
  • Zbinden G (1992). The place of in vitro methods in biomedical research. In Zbinden, G. (Ed) The Brain in Bits and Pieces, pp 11-21 , M.T.C. Verlag, Zollikon, Switzerland.

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Abstract

Des sphéroïdes peuvent être cryopréservés par des techniques qui comprennent le refroidissement par étapes. Ces sphéroïdes peuvent être formés avant la cryopréservation par rotation orbitale, selon deux ou plusieurs vitesses de rotation différentes. Il est également possible d'effectuer une rotation orbitale après la cryopréservation. La cryopréservation de sphéroïdes permet d'alimenter les hôpitaux et les laboratoires en sphéroïdes qui peuvent être dégelés et utilisés si nécessaire, ce qui évite la nécessité de les préparer in situ.
PCT/GB1998/000343 1997-02-05 1998-02-04 Preparation de spheroides et utilisation de ces derniers pour des applications therapeutiques ou diagnostiques WO1998035021A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002046374A1 (fr) * 2000-12-07 2002-06-13 University Of The West Of England, Bristol. Preparation de spheroides
JP2005514042A (ja) * 2002-01-14 2005-05-19 ユニヴァーシティ オブ ザ ウエスト オブ イングランド ブリストル 毒性試験
EP2354217A1 (fr) * 2010-02-03 2011-08-10 Universität Leipzig Dispositif de mesure et de culture intégré pour la détection sans marqueurs et la classification d'altérations cellulaires, en particulier pour la génération et la caractérisation de sphéroïdes cellulaires, composants et utilisations correspondantes
US8058443B2 (en) 2003-09-04 2011-11-15 Celgene Corporation Processes for preparing polymorphic forms of 3-(4-amino-1-oxo-1,3 dihydro-isoindol-2-YL))-piperidine-2,6-dione

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WO1997038092A1 (fr) * 1996-04-04 1997-10-16 Circe Biomedical, Inc. Nouveaux hepatocytes cryoconserves et procede de cryoconservation correspondant

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DATABASE BIOSIS BIOSCIENCES INFORMATION SERVICE, PHILADELPHIA, PA, US; SAKAI Y. ET AL.: "Functional stability of porcine hepatocyte spheroids in various culture systems under 100 percent porcine and human plasma conditions", XP002064755 *
KAAIJK P. ET AL.: "Cryopreservation of organotypic multicellular spheroids from human gliomas", NEUROPATHOLOGY AND APPLIED NEUROBIOLOGY, vol. 22, no. 6, December 1996 (1996-12-01), pages 548 - 552, XP002064881 *
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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002046374A1 (fr) * 2000-12-07 2002-06-13 University Of The West Of England, Bristol. Preparation de spheroides
JP2005514042A (ja) * 2002-01-14 2005-05-19 ユニヴァーシティ オブ ザ ウエスト オブ イングランド ブリストル 毒性試験
US8058443B2 (en) 2003-09-04 2011-11-15 Celgene Corporation Processes for preparing polymorphic forms of 3-(4-amino-1-oxo-1,3 dihydro-isoindol-2-YL))-piperidine-2,6-dione
EP2354217A1 (fr) * 2010-02-03 2011-08-10 Universität Leipzig Dispositif de mesure et de culture intégré pour la détection sans marqueurs et la classification d'altérations cellulaires, en particulier pour la génération et la caractérisation de sphéroïdes cellulaires, composants et utilisations correspondantes
WO2011095505A1 (fr) * 2010-02-03 2011-08-11 Universität Leipzig Dispositif intégré de culture et de mesure pour la détection et la classification sans étiquette d'altérations cellulaires, en particulier pour la production et la caractérisation de sphéroïdes cellulaires, composants et utilisations de ce dispositif
US20150322401A1 (en) * 2010-02-03 2015-11-12 Universität Leipzig Integrated cultivation and measurement device for label-free detection and classification of cellular alterations, in particular for generation and characterisation of cell-spheroids, components and uses thereof

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