CRYO STEM

EFFECTS OF REDUCED BACKGROUND RADIATION ENVIRONMENT ON MAINTENANCE OF CRYO-PRESERVED BIOLOGICAL MATERIAL

Introduction

Cosmic rays represent a permanent environmental factor involved both in biological evolution and cellular ageing (1). During natural selection, organisms have developed mechanisms of biological/ biochemical defence against damages caused by space radiations in the earth’s atmosphere. It is interesting to notice that different organisms and cells exposed to a small dose of radiations or chemical mutagenic agents can become resistant to subsequent radiation dose expositions (adaptive response) (2). This cell adaptive response to cosmic rays exposition has been recently studied at Gran Sasso National Laboratories (LNGS). LNGS are provided with an underground laboratory that offers the opportunity to evaluate the effects of a low radioactivity environment on various organisms. Biological experiments, performed in the LNGS (Pulex and Pulex-2 projects) on yeasts (3) and on chinese hamster fibroblasts (4), suggest that the background radiation has an important role in determining various cellular adaptive processes. In particular, these experiments show that cells grown at a low dose of radiation: 1) are less protected from DNA damages induced by chemical and physical agents; 2) are more sensitive to undergo apoptosis; 3) show a different behaviour in defending them selves against oxidant agents. Studies performed in space, where cosmic rays are greater than those on the earth’s surface, have allowed to highlight the main cellular targets and the possible consequences caused by cosmic radiation. These experiments have demonstrated that cosmic radiations increase: DNA strand breaks, induced mutations (5-7), lipidic peroxidation (8), cytokine secretion (9), but they also induce inhibition of proliferation, cell differentiation, cell-cell interaction and cytotoxic activity mediated by the immune system (5,9). Moreover, it has been observed that cosmic rays induce expression of p53, a protein that, causing apoptosis or blocking G1/S transition of cell cycle, safeguards genomic stability and protects from tumour formation induced by chemical and physical agents (5,6,10). In fact, radiation insult can induce DNA strand breaks and mutations that, depending both on damage extent and cell ability to repair it, can finally produce: 1. cell cycle arrest to allow DNA repair (usually p53-dependent); 2. chromosomal translocation or deletion (when DNA is badly repaired) that can favour the generation of tumour cells (usually when the p53 protein does not work); 3. programmed cell death (apoptosis) when the amount of damage is too important to be repaired without the risk of generating tumour cells (usually p53-dependent). Summarizing, even if cosmic rays induce the enzymes able to protect the cells from radiation damages, they also promote the generation of tumour cells and cell ageing. On the other hand, an expedient to slow down the ageing process of cells or organisms (that have to be maintained unaltered for a long time) utilizes low temperature. Cryopreservation of cells, tissues or organisms (by mean of freezing, vitrification or hibernation) can reach in some cases the halt of the metabolic activities (below -130 °C or 143 °K) and the storage in liquid nitrogen (- 196 °C or 77 °K) or in cold nitrogen vapor (- 150 °C or 123 °K) offers the most secure form of preservation. However, it is important to consider that cryopreserved biological material cannot repair the damages produced by background radiations, therefore an accumulation of this alterations could occur over time. It could be therefore possible that the cryopreserved cells after defrosting show the typical aspects of irradiated cells (cell cycle arrest, apoptosis, and cancerogenesis), in particular if they has been stored for a long time period (decenniums). It is, in fact, a common experience that the percentage of viable cells slowly tends to decrease over time in defrosted cryo-preserved cells (11), in this case to evaluate the whole death process (and not only the rapid necrotic one) it is important to culture the cells at least overnight. To this regard, it has to be remembered that while necrotic cell death is a rapid and passive cell lysis due to a strong environmental insult (bad freezing or defrosting, chemical effects of cryoprotectants such as DMSO), apoptotic cell death is an active and slow cell process (requiring hours or days) regulated by a cell homeostatic control that eliminates cells potentially dangerous to its organism (i.e. damaged cells that may become tumour cells). Informations from cryocrystallography in which biological macromolecules are subjected to radiations for determining their tridimensional structure, suggest that very low temperatures (100 °K) slowing down the diffusion of free radicals produced by incident radiations, considerably reduce (even if not completely block) damages induced by X-rays (12). It is interesting that red cell viability does not seem to be affected by gamma radiations (2500 cGy or 4000 cGy) (13). Altogether these preliminary remarks suggest that, a reduced background radiation could favour the maintenance of frozen cells, and thus their vitality and cell cycle restart (phenomena related with DNA strand breaks caused by cosmic rays) after thawing. On the other hand, while cosmic radiation influence positively the induction of defensive systems against radiation and other mutagenic agents in living cells, the low background radiation environment at the LNGS underground laboratory should not negatively affect the enzymatic systems of cryo-preserved cells. Although a lot of experiments have demonstrated that cosmic radiations can induce cell death, tumors and ageing in living cells, the question that we put is: after a long period of storage, are these same effects also induced in cryopreserved cells?

Aim of the study
The main purposes of this research are to evaluate after thawing: A) the differences of vitality (% of living cells) and other cell functions integrity of cryo-preserved cells (in particular of haematopoietic stem cells) in the presence of normal or reduced background radiations; B) the differences of vitality and other cell functions of anucleated (with no DNA) cryo-preserved cells (in particular of erythrocytes) in the presence of normal or reduced background radiations. This model will allow to evaluate the effects on membrane lipid peroxidation and protein alteration induced by background radiations, without the most important effect that occurs at DNA level. After thawing, vitality and cell integrity of cryo-preserved will be evaluated analysing some cellular parameters, such as: 1) cell mortality (14,17); 2) cell cycle and apoptosis (14,16-21); 3) surface antigen expression, particularly focusing on stem cell molecules and adhesion molecules regulating cell-cell interactions (22-26); 4) cytokine production (27); 5) expression of intracytoplasmic molecules such as Bcl-2 and p53 (10); 6) DNA repair capability from damages induced by chemical and physical agents (14, 16-18, 28,29). The aim of this study is to evaluate whether, in the LNGS underground laboratory, there are advantages in conserving biological material and in particular hematopoietic stem/progenitor cells (CD34+) from cord blood or other sources. Nowadays, it is well known that stem cell transplants, necessary in some pathologies, need a high level of compatibility between donor and host. To reach this goal numerous bank of bone marrow and umbilical cord blood have been constituted. The research of a compatible donor, in the case of allogenic transplantation, may take a lot of time; on the other hand, in the case of autologous transplantation, which means with stem cells belonging to the transplanted subject himself (from umbilical cord blood or peripheral blood), stem cells have to be previously withdrawn and sometimes maintained integral for a long period. Having viability of CD34+ (hematopoietic stem/progenitor) cells important effects on the subsequent hematopoietic engraftment, we believe that it is fundamental, not only the quality of the freezing process, but also the environment in which cells are preserved. The identification of an environment that allows the maintenance of frozen biological material (in particular of stem cells of which cord blood is relatively rich) for long periods (also decades) without having large cellular alterations after thawing, it would be very important for the organization of a long lasting stem cell bank. Such environment could be that, with reduced background radiation, of Gran Sasso underground laboratory. These kind of studies could also bring light on the state of deterioration and on the safety of numerous hospital vials or bags stored for years frozen and never used. At the same time, to obtain theoretical data about the effects of background radiations, we will perform experiments on cryopreserved cells treated with radioactive sources that could simulate background radiations.

Materials And Methods
We will use: A) different leukaemic lines, K562 (radiation resistant), Jurkat (radiation sensitive), HL-60 (radiation sensitive, p53 negative); B) haemotopoietic primary cells from peripheral blood and cord blood samples. First, the cells will be expanded and frozen at -80 °C (or 193 °K) at the lab of Centro di Citometria e Citomorfologia of Urbino. Briefly, cells will be resuspended in a freezing solutions (50% FBS, DMSO 10%, 40% RPMI 1640), stocked into 2 ml vials with 5-50x10⁶ cells/ml and frozen at -80 °C. Then the cells will be carried to LNGS in dry ice (-78,5 °C or 194,5 °K), and subsequently maintained in dewars with liquid nitrogen (GT38, Air Liquide, Paris, France), either in the LNGS underground laboratory (Special Technique Service) in low background radiation conditions either in the LNGS outside laboratory (Chemistry Service) in normal background radiation environment. Every 2 months, both cell groups will be carried to the outside laboratory (Chemistry Service) in dry ice and rapidly thawed in water bath at 37 °C (or 310 °K). To minimize the sources of variability, experiments will be performed in triplicates by the same operator and the quantity of radiations, present in the two laboratories, will be periodically tested. After thawing, cells from both laboratories will be analysed to evaluate: - cell mortality, soon after thawing and after an overnight incubation (37 °C, 5% CO₂, necessary to evaluate apoptosis), by mean of trypan blue coloration and cellular count with Neubauer; - DNA strand break evaluation by TUNEL in situ or in situ nick translation (14, 15); - Cell cycle kinetics after thawing by Propidium Iodide and/or Bromodeoxyuridine incorporation (14-16); - apoptotic process and cell death, soon after thawing and after an overnight incubation (37 °C, 5% CO₂, necessary to evaluate apoptosis), by flow cytometric analysis (ethanol/propidium iodide or, if possible, annexin-V/propidium iodide (14, 17,18)], DNA analysis by elettrophoresis (ladder) and TUNEL (14, 15), and morphological analysis by TEM; - cell cycle and the protein correlated [in particular p53 (10,14, 17,18)]; - surface antigen expression, with particular attention to stem cell molecules (CD34, CD133, CD90, CD135, CD117) and adhesion molecules in particular stem cell antigens some cytokines and the adhesion molecules (CD11a, CD11b, CD11c, CD2, CD54) (22-26); - intracellular cytokine production (27); - ability to repair DNA damages induced by chemical (camptothecin) and physical (ultraviolet rays) agents by mean of cell cycle analysis in flow cytometry (14, 16-18, 28,29); - Bcl-2 expression and, if it is useful, cell membrane peroxidative state using fluorescent markers in flow cytometry (10). The experiment that we propose to perform in underground LNGS lab is simple and well verifiable. In fact, different aliquots of the same sample of cells will be analysed by the same operator using same reagents and same instrumental analysis. For what concern the part of experiment that have to be performed in the underground LNGS lab, a very limited space will be required (the dimension of the GT38 dewar, see Fig. 1) because, in that lab, biohazard material will not be manipulated. Fig.1 Dewar GT38, Air Liquide, Paris, France

Fig.1 Dewar GT38, Air Liquide, Paris, France

Personnel and equipment required

A ) The personnel required for the evaluation of cryo-preserved cells has to be properly trained to cell culture techniques (cell culture and cell expansion, freezing and thawing) and in case to flow cytometric analysis of samples.

The following equipment and reagents are necessary:

• 1 inverted microscope for cell observation;

• 1 laminar flow hood for cell manipulation (splitting and freezing) in sterility;

• 1 CO 2 incubator for cell expansion before freezing;

• 1 thermostated bath for cell thawing (37° C);

• 1 freezer -80° C for cell gradual (initial) freezing;

• 2 dewars (GT38, Air Liquide, Paris, France) for cell cryo-maintenance; one in each laboratory (outside and underground laboratories);

• 1 bench centrifuge for washes;

• 1 mobile flow cytometer able to perform cell absolute counting (CyFLow Fig.2 , gently provided by Partec, Munster, Germany);

• 1 Neubauer's chamber for cell counting;

• material for cell cultures (sterile flasks, medium, FBS, cryo-vials, DMSO, antibiotics, glutamine, ecc.);

• various material to perform flow cytometric analysis (specific antibodies and fluorescent dies), optical fluorescent microscopy (nucleotides and kit TUNEL), and electronic microscopy (fixatives and material for inclusions).

The possibility to have a small bench cytometer for flow cytometric analysis, will allow to perform analyses directly in the LNGS outside laboratory. However, in most of the cases, cells could be transported and analyzed at the Cytometry and Cytomorphology Centre of Urbino. The cell stabilisation with TRANSFIX® will allow surface antigen analysis ( 30,31 ), while cell freezing or cell fixation with formaldehyde, ethanol or glutaraldehyde will allow DNA electrophoretic evaluation (ladder), analysis of intracytoplasmic proteins, cell cycle, DNA (TUNEL) and cell morphology (TEM), respectively.

For this reason, a permanent work at the LNGS underground laboratory, will not be necessary and thus most of the activities will be performed at the LNGS outside laboratory or at the Cytometry and Cytomorphology Centre in Urbino. This latter is a reference centre at an international level for the flow cytometric evaluation of the cell mortality and for the analysis of the haematopoietic stem cells (see pubblications 14-30 ).

Fig.2 Mobile flow cytometer able to perform cell absolute counting (CyFLow , Partec, Germany)

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