Effect of cryopreservation on viability and growth efficiency of stromal-epithelial cells derived from neonatal human thymus

Accepted Manuscript Effect of cryopreservation on viability and growth efficiency of stromal-epithelial cells derived from neonatal human thymus Valentin P. Shichkin, Oleksandr I. Gorbach, Olga A. Zuieva, Nataliia I. Grabchenko, Irina A. Aksyonova, Boris M. Todurov PII:

S0011-2240(17)30122-0

DOI:

10.1016/j.cryobiol.2017.06.010

Reference:

YCRYO 3862

To appear in:

Cryobiology

Received Date: 30 March 2017 Revised Date:

26 June 2017

Accepted Date: 27 June 2017

Please cite this article as: V.P. Shichkin, O.I. Gorbach, O.A. Zuieva, N.I. Grabchenko, I.A. Aksyonova, B.M. Todurov, Effect of cryopreservation on viability and growth efficiency of stromal-epithelial cells derived from neonatal human thymus, Cryobiology (2017), doi: 10.1016/j.cryobiol.2017.06.010. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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ACCEPTED MANUSCRIPT Effect of cryopreservation on viability and growth efficiency of stromal-

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epithelial cells derived from neonatal human thymus

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Valentin P. Shichkin a, *, Oleksandr I. Gorbach a, Olga A. Zuieva a, Nataliia I. Grabchenko a, Irina

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A. Aksyonova b, Boris M. Todurov b

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a

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Human Development “Ukraine”, Kyiv 03115, Ukraine

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b

Research Centre of Immunology and Biomedical Technologies, Open International University of

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Institute of Heart, Ministry of Health of Ukraine, Kyiv 02660, Ukraine

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________________________________ Abbreviations: 7-AAD, 7-aminoactinomycin D; annexin V-EGFP, annexin V-enhanced green

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fluorescent protein; Abs, antibodies; CPM, cryoprotective medium; CM, culture medium; FBS,

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fetal bovine serum; FSP, fibroblast surface protein; FACS, fluorescein activated cell sorter; PI,

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propidium iodide; SCB, Stem-CellBanker; TESC, thymic epithelial stem cells.

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* Corresponding author. E-mail address: [email protected] (V. Shichkin).

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Abstract

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The thymus is the major site of T lymphocyte generation and so is critical for a functional

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adaptive immune system. Since, thymectomy is a component of neonatal surgery for congenital

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heart diseases, it provides great potential for collection and storage of thymic tissue for autologous

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transplantation. However, specific investigation into the optimum parameters for thymic tissue

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cryopreservation have not been conducted. In this research, we evaluated the effect of different

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cryoprotective media compositions, which included penetrating (Me2SO, glycerol) and non-

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penetrating (dextran-40, sucrose, hydroxyethyl starch) components, on the viability and

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functionality of frozen-thawed human thymic samples to select an optimal cryoprotective medium

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suitable for long-term storage of thymic tissue and a stromal-epithelial enriched population. Our

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primary focus was on receiving, low-temperature storage, culturing and evaluation of thymic tissue

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samples from newborns and infants with congenital heart diseases, who had undergone thymectomy

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as a part of standard surgical procedure. Thus, this work builds the platform for autologous clinical

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intervention into the thymus-deficient patients with congenital heart diseases. From our data, we

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conclude that although there were no significant differences in efficiency of tested cryoprotective

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media compositions, the combination of Me2SO and dextran-40 compounds was the most suitable

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for long-term storage both thymic cell suspensions and thymic fragments based on the viability of

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CD326+ epithelial cells and stromal-epithelial cell monolayer formation.

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Keywords: Cryopreservation, Liquid nitrogen, Long-term storage, Human thymus, Stromalepithelial cell cultures, Cell phenotype 47 48 49 50

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1. Introduction

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The thymus is one of the central organs of immune system; it is the major site of T lymphocyte

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generation and hence is critically important for a functional adaptive immune system. However,

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with aging, the thymus is among the first organs to degenerate in normal healthy individuals [12].

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Thymus function is suppressed under stress and by influence of various cytotoxic effects, including

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some medical treatments [36]. While many functions of the adaptive immune system are established

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during infanthood, partial or total thymectomy is a part of some surgical procedures, particular at

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the congenital heart diseases, especially in newborns [5]. Some forms of severe combined

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immunodeficiency such as DiGeorge syndrome, Nezelof syndrome, Louis-Bar syndrome, Swiss

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syndrome are due to inherent thymic aplasia [8, 20, 32]. Impaired thymic function has a number of

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consequences for the immune system including increased susceptibility to infections and

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autoimmunity, high risk of cancer development as well as decreased response to vaccines with age

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[1, 10, 17, 22, 29, 35]. Because of these, there is currently a great interest in developing strategies

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for boosting thymic function by either cell replacement or regenerative strategies. In this regard,

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the regulation of thymic recovery is extremely relevant and clinically significant for immune system

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rehabilitation especially in thymectomized newborns and infants.

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Since, thymectomy is a component of neonatal surgery for congenital heart diseases [16, 19; 30],

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it provides great potential for collection and storage of thymic tissue and its use for autologous

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transplantation. Thus, development of validated protocols for storage and autologous transplantation

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of thymic tissue or stromal-epithelial cells, primarily thymic epithelial cells (TEC) can lead to the

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successful reconstitution of immune system functions as was shown on animal models and in some

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clinical investigations [3; 4, 9, 15, 23, 24, 27, 28]. However, while the born marrow and cord blood

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are priority sources of hematopoietic stem cells, and cryopreservation protocols for these tissues

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were established, as have been protocols for cryopreservation of stromal cell preparations from

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adipose tissue and from bone marrow [2, 7, 13, 33, 37, 38], the thymus as a potential source for

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stem cell therapy has received little attention. There are only a few reports of freezing thymus

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tissue, where protocols with dimethyl sulfoxide (Me2SO) and fetal bovine serum (FBS) were used

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for cryopreservation of thymic tissue or thymic stromal cells [6, 18, 25]. No special investigations

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have been performed concerning thymic tissue cryopreservation. In current research, we have

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evaluated the effect of different cryoprotective media (CPM) compositions, which included

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penetrating (Me2SO, glycerol) and non-penetrating (dextran-40, sucrose, hydroxyethyl starch)

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components [21] on the viability and functionality of frozen-thawed human thymic samples to

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select an optimal cryoprotective medium suitable for long-term storage of thymic tissue and

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stromal-epithelial population enriched by TEC prepared by different methods. Our primary focus

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was on thymic tissue samples obtained from newborns and infants with inherent heart diseases. Our

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data indicate that although there were no significant differences in efficiency of tested

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cryoprotective media compositions, the combination of Me2SO and dextran-40 compounds was the

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most suitable for long-term storage both thymic cell suspensions and thymic fragments based on the

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viability of CD326+ epithelial cells and stromal-epithelial cell monolayer formation.

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ACCEPTED MANUSCRIPT 2. Materials and methods

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2.1. Thymus collection and evaluation

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Human thymic samples were obtained from newborns and children under the age of 3 years as a

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part of standard operation procedure on the heart from patients with congenital heart diseases.

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Thymus sampling was performed under aseptic conditions in the Institute of Heart of the Ministry

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of Health of Ukraine at the Pediatric Department (Kyiv, Ukraine). Informed consents from the

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parents for thymic tissue collection and using for research aims within ThymiStem project were

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received before surgery and approved by Bioethics Committees of the Institute of Heart and the

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University “Ukraine”. According to the regulations of the Ministry of Health of Ukraine, before the

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surgical operation, patients were tested for Hepatitis B, Hepatitis C, Treponema Pallidum and HIV

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infections. Only thymic samples from patients free of these infections were collected for further

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research.

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For transportation, thymic lobes were put into 100 ml sterile plastic tubes for tissue sampling

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with 25 ml of RPMI-1640 (Sigma, USA) or DPBS (ThermoFisher Scientific, USA) with antibiotics

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(streptomycin/penicillin) in concentration of 50 U/ml of penicillin and 50 µg/ml (33.55 IU) of

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streptomycin (both from ThermoFisher Scientific, USA) – transportation medium. The tubes were

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transported in an isothermal box on ice to the laboratory. Total time, including thymus removal and

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transportation to the laboratory, was about two hours. Before preparation and freezing, thymic lobes

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were weighed in sterile conditions, and ranged from 3.15 g to 74.56 g (mean, 24.41 g). Eighty

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thymuses were collected, prepared and stored in liquid nitrogen from several days up to two years

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with use of different CPM compositions. Thymic samples were analyzed with a set of methods as

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described below.

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2.2. Thymic tissue preparation

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Thymic lobes were transferred from the transportation tubes into large Petri dishes (d = 10 cm,

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ABC Scientific, USA) with RPMI-1640 (Sigma, USA) or DPBS (ThermoFisher Scientific)

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transportation medium, and cut into fragments approximately of 0.8 – 1.0 cm3 in size using sterile

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scissors. Cell suspensions were obtained where required by mechanical disaggregation or enzymatic

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digestion of thymic fragments, performed as described below.

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For mechanical disaggregation, thymic fragments were homogenized in a transportation medium

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using a glass Dounce mechanical homogenizer, filtered through a sterile nylon filter (Falcon® 70

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µm Cell Strainer, Fisher Scientific, USA) and washed 3 times in a transportation medium by

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centrifugation at 300 g for 5 min. All procedures were performed on ice.

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Enzymatic disaggregation was performed as described [14, 26, 34] with some modifications for

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human thymic tissue. Thymic lobes were cleaned from capsule and other non-thymic tissues under

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aseptic conditions. Then they were transferred into a Petri dish in transportation medium and cut

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with sterile scissors into 0.8 – 1 cm3 pieces. These pieces were gently agitated to release T cells.

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Each thymic piece was then cut into as many small pieces as possible and agitated gently in a Petri

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dish with transportation medium to release remained T cells. For further removal of remaining T

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cells, thymic pieces were transferred into 50 ml centrifuge tubes containing 50 ml of transportation

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medium and agitated at room temperature for 30 min. The supernatant containing thymocytes was

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removed, and the remaining thymic pieces transferred to a conical tube containing 10 ml of

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transportation medium. The thymic fragments were pipetted and, after the thymic pieces had settled,

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supernatant was removed. This procedure was repeated two further times.

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For further enzymatic isolation of thymic stromal-epithelial cells from these primary treated

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thymic fragments, the thymic pieces were incubated for 30 min at 37°C in 5 ml of transportation

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medium with collagenase IV (0.125% w/v; Gibco, USA) and DNAse I (0.1% w/v; Gibco, USA)

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gently agitating with a pipette every 5 min. After the thymic pieces had settled, the supernatant that

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contained stromal-epithelial cells, was collected into a new 50 ml centrifuge tube with 50 ml of

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transportation medium, and was kept in CO2 incubator until use. Fresh collagenase IV with DNAse

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I in transportation medium was then added to the tube containing the residual thymic pieces, and the

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incubation with agitation was repeated as described above. After the thymic pieces had settled,

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supernatant containing released stromal-epithelial cells was collected again, mixed with the cell

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portion collected before and centrifuged at 300 g for 5 min. The cells were resuspended in

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Ca++/Mg++- free DPBS with 5% FBS and 0.1% DNAse I (FACS buffer), centrifuged at 300 g for 5

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min to pellet the cells. Finally, the cells were resuspended in the required cell culture medium, and

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the cell number and cell viability were evaluated. An aliquot of cells was resuspended in fresh

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FACS buffer for analysis of primary thymic cell composition by flow cytometry.

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2.3. Thymic samples freezing and thawing procedures

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The freezing procedures were carried out on thymic fragments (0.8 – 1 cm3 in size), thymic cell

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suspensions after mechanical homogenization, thymic cell suspensions after enzymatic digestion,

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and cultured stromal-epithelial cell suspensions. Thymic samples were placed into 2 ml round

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bottom cryovials (Greiner Bio-One, Germany) with 1ml of a cryoprotective medium (CPM). All

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procedures were performed on ice. The cryovials were then moved into a -80° C freezer in a

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styrofoam box to provide gradual temperature drop (from a room temperature to -80° C), and the

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next day, were moved into Dewars with liquid nitrogen for long-term storage at -196° C. Fresh

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prepared cell suspensions that contained about 90% of thymocytes and 10% of stromal-epithelial

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cells were frozen in concentration 1 – 5 × 107 cells/ml in 1 ml of CPM to obtain finally about 1 – 5

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× 106 of stromal-epithelial cells per 1 ml. Cultures of stromal-epithelial cells free of thymocytes

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were frozen in concentration 1 – 5 × 106 cells/ml in 1 ml of CPM. In these ranges of concentrations

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the fluctuations of the cell viability were insufficient as it was estimated in our preliminary

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experiments. We tested nine different CPM compositions, which included penetrating (Me2SO and

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glycerol) and non-penetrating (dextran-40, sucrose and hydroxyethyl starch) components in the

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proportions as showed in Table 1. Commercial cryoprotective medium Stem-CellBanker (SCB)

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(AMS Biotechnology, UK) was used in some experiments for comparison. Frozen cryovials with thymic cell suspension or thymic fragments were put into a water bath at

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37° C for 2 – 3 min. Cell suspensions were transferred to 15 ml tube and washed 3 times in 10 ml of

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FACS buffer by centrifugation at 400 g for 5 min. Thymic fragments after thawing were transferred

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into Petri dish (d = 10 cm) and washed twice in 10 ml of DPBS transportation medium, and than

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prepared by enzymatic digestion or mechanical homogenization to get cell suspension as above

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(section 2.2).

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Table 1

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Cryoprotective media compositions tested.

Medium composition

CPM1

90% FBS + 10% Me2SO

CPM2

50% FBS + 10% Me2SO + 40% RPMI-1640

CPM5 CPM6 CPM7

SCB

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15% glycerol + 85% RPMI-1640 15% glycerol + 85% 0.1 M sucrose solved in RPMI-1640

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CPM8

0.7 M Me2SO + 0.1 M sucrose solved in DPBS

95% of 6% solution of hydroxyethyl starch + 5% Me2SO

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Cryoprotective medium

CPM3

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95 % of 5 % solution of polyglucinum (dextran-40) + 5% Me2SO 5% solution of dextran-40

Serum-free, Me2SO-containing

2.4. Thymic culture preparation

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Collected thymuses were used for tissue and cell culture preparation in four different ways. Big

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fragments about 0.8 – 1 cm3 in size were placed into Petri dishes (d = 6 cm) (one fragment per dish)

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with 4 ml of FBS-containing RPMI-1640 culture medium (CM-1) or in serum-free culture medium

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(CM-2), and fragments were cultured until monolayer formation of stromal-epithelial cells was

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observed. CM-1 and CM-2 compositions were as described below. Some of the thymic fragments

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were cut into smaller pieces (1 – 2 mm), and 5 – 6 such explants were seeded into small Petri dishes

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(d = 3 cm) as described above. Some of the thymic fragments were homogenized in RPMI-1640

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using a Dounce mechanical homogenizer. Then cells were filtered through a sterile nylon filter, and

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cell suspensions were seeded into small Petri dishes in CM-1 or CM-2 at a cell concentration of 20

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× 106 per ml. Some of the thymic fragments were enzymatically digested, and cells were seeded

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into small Petri dishes as described above.

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CM-1 composition was as described by Markert et al [24] with some modifications. We used

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RPMI-1640 (Sigma, USA) with 10% or 5% FBS (Gibco, USA), 25 mM HEPES (Sigma, USA), 2

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mM L-glutamine (ThermoFisher Scientific, USA), 50 U/ml penicillin (ThermoFisher Scientific,

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USA), and 50 µg/ml (33.55 IU(s)) streptomycin (ThermoFisher Scientific, USA).

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CM-2 composition was as described by Ropke [31] with modifications. We used DMEM/ Ham’s

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F12 (1:1) with low calcium (0.021 mg/ml) (Gibco, USA) with addition of 20 ng/ml epidermal

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growth factor (Gibco, USA), 0.5 µg/ml hydrocortisone (Sigma, USA), 3 µg/ml insulin (Sigma,

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USA), 1x RPMI amino acids solution (Sigma, USA), 2 mM L-glutamine (ThermoFisher Scientific,

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USA), 250 U/ml penicillin (ThermoFisher Scientific, USA), 25 µg/ml streptomycin (ThermoFisher

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Scientific, USA).

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2.5. Cell viability evaluation by microscopy

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Cell viability was measured with trypan blue and/or annexin V-EGFP/ethidium bromide

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staining. For trypan blue staining, cell suspension was prepared approximately in concentration of 1

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× 106 cells/ml, and 20 µl of cell suspension was gently mixed with equivalent value of 0.4% trypan

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blue solution (Sigma, USA). After 3 - 5 min staining, 10 µl of cell suspension was put into a

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hemocytomet er (Goryaev chamber) and the percentage of viable cells was counted using light

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microscopy. For annexin V-EGFP/ethidium bromide staining, cells were centrifuged at 300 g for 5 min and

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the medium was discarded. The cells were resuspended in 1x DPBS binding buffer that was

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prepared from 10x stock (0.1 M HEPES pH 7.4, 1.4 M NaCl, 25 mM CaCl2). 1 - 2 µg of annexin V-

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enhanced green fluorescent protein (annexin V-EGFP) (BeckmanCoulter, France; 2 mg/ml) and 1µl

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of ethidium bromide (Sigma, USA) from a 10 mg/ml stock solution was added to 1 x 106 cells/ml.

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The cells were incubated for 15 min at the room temperature and visualized using fluorescence

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microscopy at 470 nm (annexin V-EGFP) and 540 nm (ethidium bromide) and Fiji image analysis

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software (http://fiji.sc).

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2.6. Light and fluorescent microscopy assay

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Visual identification and imaging of cell growth in cell cultures were performed with an inverted

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fluorescence microscope (Carl Zeiss Axio Vert. A1). Image processing was performed using ZEN

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light edition 2011 program.

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2.7. Flow cytometry cell analysis

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Flow cytometry analysis (FACSCalibur, Becton Dickinson, USA) was used for evaluation of the

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percentage of cells undergoing apoptosis or secondary necrosis by staining with annexin V-

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EGFP/propidium iodide (annexin V-EGFP/PI) (BeckmanCoulter, France), detection of dead cells

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by staining with PI and 7-aminoactinomycin D (7-AAD) (Sigma, USA) as well as for evaluation of

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the thymic cell phenotype by staining with antibodies (Abs) to CD326/EPCAM and to CD45

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antigens (PerCP/Cy5.5-labeled mouse IgG2bκ and IgG1κ, respectively) and to STRO-1 antigen

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(FITC-labeled mouse IgMλ) (Biolegend, USA), and to fibroblast surface protein (FSP) (PE-labeled

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recombinant human IgG1) (Miltenyi Biotec, Germany). Cell staining was conducted in accordance

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with the manufacturer's recommendations. In brief, 105 cells in 100 µl of FACS buffer and 5 µl of

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antibody or other labeled reagent were added into each tube. All procedures were performed on ice.

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Samples were incubated for 30 min at 4 - 8°С, washed with 1 ml of FACS buffer and centrifuged at

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300 g for 5 min at the room temperature. The supernatant was removed, and 500 µl of FACS buffer

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were added into each tube. Samples were kept at 4 - 8°С in the dark until analysis.

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2.8. Thymic samples preparation for flow cytometry analysis

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For thymic cell cultures analysis, supernatant was fully removed from a culture flask (25 cm3)

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and 2 ml of 0.5% trypsin with 0.2% EDTA (Sigma, USA) warmed to 37°C were added to the flask

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for 3 min. To inactivate trypsin-EDTA, 5 ml of FACS buffer were added to the flask, and cells were

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gently resuspended. Cell suspensions were transferred into 15 ml tubes and centrifuged at 300 g for

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5 min. The supernatant was aspirated. Cells were then resuspended and counted using a

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hemocytometer; flow cytometry analysis was as described above.

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For analysis of frozen thymic cell samples, the cells were thawed in water bath at 37°C for 2 - 3

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min and washed in 10 – 15 ml of FACS buffer by centrifugation at 300 g for 5 min at the room

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temperature. The cells were resuspended in 1 ml of FACS buffer and, if clumps were observed,

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transferred into a 50 ml tube through a 70-µm cell strainer. In order to make sure no cells are left

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behind on the strainer, 2 ml of FACS buffer was added. The cells were centrifuged at 300 g for

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5 min at the room temperature. If clumps were observed again, a previous step with a cell strainer

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was repeated.

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2.9. Adhesive cell cultures confluency evaluation

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Confluence is commonly used to estimate adherent cell number in a culture dish or flask,

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referring to the proportion of the surface, which is covered by cells. For confluence evaluation in

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the thymic stromal-epithelial cell cultures, we used a 10-grade scale with 10-percentage steps. One

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grade was equivalent to 10% of the dish or flask surface covered by cells and 10 equivalent to 100%

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of the surface. Visual identification, confluency evaluation and imaging of cell growth in cell

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cultures were performed with an inverted fluorescence microscope (Carl Zeiss Axio Vert. A1).

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2.10. Statistics

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Experimental data were processed by means of variation statistics. Mean and standard deviation

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(SD) were used. To compare the parametric data in two groups, t-test was used. When р < 0.05 data

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were considered as statistically significant.

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3. Results

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3.1. Effect of cryoprotective media composition and samples preparation method on total thymic

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cell viability

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Because no special study was performed concerning thymic tissue cryopreservation, we have

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primarily chosen eight compositions of cryoprotective media to compare their protective effects on

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cell viability in thymic fragments (the most fast and harmless samples preparation method) in

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dependence on cell samples preparation method evaluated after thawing. The criterion for

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evaluation of the most appropriate cryoprotective medium and cell samples preparation method was

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the percentage of viable cells assessed by trypan blue and annexin V-EGFP/ethidium bromide

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staining in thymic fragments after their thawing and next cell suspension preparation or in frozen

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cell suspensions immediately after thawing. A further criterion for assessment of the cell viability in

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frozen thymic samples was their ability to form a monolayer of stromal-epithelial cells and to

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establish long-term cultures of these cells. As shown in Table 1, no significant difference in

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protective efficiency of CPM1, which contained 90% FBS and CPM2, which contained 50% FBS,

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was observed. In further experiments, we used CPM-1 composition as a standard control compared

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to other CPM compositions. In subsequent experiments, it was important to estimate the protective

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efficiency of the different CPM, that contained penetrating cryoprotectants (Me2SO, glycerol) and

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non-penetrating cryoprotectants (sucrose, dextran-40, hydroxyethyl starch) as this relevant to future

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clinical application. After thawing of thymic samples stored in CPM with glycerol (CPM4) and

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glycerol + sucrose (CPM5), the number of viable cells was significantly lower than in media

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containing Me2SO + dextran-40 (CMP7) or Me2SO + hydroxyethyl starch (CMP6). The number of

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viable cells in thymic samples frozen in CPM6 and CMP7 were in a range of 87.4 – 98.2%. The

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number of viable cells frozen in a medium with only dextran-40 (CPM8) was lower (65.4 – 84.4%)

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but still higher than in CPM1 and CPM2. Thus, CPM7 and CPM6 showed the highest cell viability

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rate in all thymic samples after freezing, while CPM5, CPM4 and CPM3 showed the lowest

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viability rates. The percentage of viable cells was statistically significant compared to CPM1 for of

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these most CPM (Table 2). We have not found significant influence of cell samples preparation

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method (small explant culture, mechanical homogenization, enzymatic digestion) on cell viability

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using CPM3, CPM4, CPM5, CPM6, CPM7 and CPM8 for cryopreservation of thymic fragments.

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However, cell viability was significantly lower in cell samples prepared by enzymatic digestion of

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thymic fragments stored in CPM1 and CPM2 that contained FBS.

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From these data, we concluded that the most suitable cryoprotective medium for freezing of

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thymic tissue was the combination of dextran-40 and Me2SO (CPM7) or the combination of

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hydroxyethyl starch and Me2SO (CPM6). These combinations are also preferable for future clinical

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application of stored thymic tissue, because they contain relatively low concentrations of Me2SO

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and do not contain FBS. Overall, the presence of 5% of the penetrative cryoprotectant Me2SO in a

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cryoprotective media significantly increased cell viability in frozen thymic samples.

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Table 2

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Effect of cryoprotective media composition and cells preparation method on cell viability in frozen-

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thawed thymic fragments evaluated by staining with trypan blue.

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Cryoprotective media

CPM1

†

Cell viability obtained

Cell viability obtained

Cell viability obtained

after freezing of thymic

after freezing of thymic

after freezing of thymic

fragments followed by

fragments followed by

fragments followed by

thawing and explant

thawing and mechanical

thawing and enzymatic

culture (%)

homogenization (%)

digestion (%)

77.9 ± 7.1

65.0 ± 3.0

48.3 ± 2.2

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63.0 ± 5.2

**37.2 ± 3.0

CPM3

*48.3 ± 3.0

47.8 ± 6.0

**30.2 ± 4.6

CPM4

**32.8 ± 2.4

**30.8 ± 2.4

**32.4 ± 3.6

CPM5

**30.3 ± 1.2

**32.4 ± 4.0

**28.7 ± 4.0

CPM6

94.8 ± 8.0

**87.4 ± 2.2

**94.3 ± 1.2

CPM7

97.5 ± 4.3

**90.2 ± 3.0

CPM8

75.3 ± 6.6

65.4 ± 3.5

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CPM2

**98.2 ± 1.2 **84.4 ± 8.7

Thymic fragments were stored in liquid nitrogen for 30 days, and 5 randomly selected samples for

349

each CPM composition from the same thymus were prepared by mechanical homogenization or

350

enzymatic digestion after thawing, and cell viability was evaluated with Trypan blue staining. †After

351

thawing, each thymic fragment was cut into small explants (1 - 2 mm), and 5 explants were seeded

352

in small Petri dishes (d = 3 cm) in 2.5 ml of CM-1 and cultured for 24 h. Cells released from these

353

explants were collected and analyzed by light microscopy. Data show mean ± SD for n = 5. *p <

354

0.05 and **p < 0.01 compared to samples frozen in CPM1.

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355

Since trypan blue staining does not enable differentiation of apoptotic and necrotic cells, the

357

thymic cell suspensions were also stained with annexin V-EGFP/ethidium bromide directly after

358

thawing. Visual assessment of the samples was performed using fluorescence microscopy (Fig. 1).

359

Analysis with annexin V-EGFP/ethidium bromide double staining revealed that the cell suspension

360

contained primarily double stained cells. Double staining indicates a violation of the cell integrity

361

that may occur as a result of necrosis, which can be a result of mechanical damages of the cells as

362

well as follow the freezing-thawing procedure of thymic samples.

363

Fig. 1.

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364

For further experiments, the most promising cryoprotective medium CPM7 was selected and

365

compared to cryoprotective medium CPM1. Assessment of the efficacy of the cryoprotective

366

compositions was performed after enzymatic digestion of the non-frozen thymic fragments (0.8 -

16

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1.0 cm3) and subsequent freezing of cell suspensions in selected CPM as well as for frozen-thawed

368

thymic fragments (0.8 - 1.0 cm3). After the freezing-and-thawing procedure, the cell viability rate

369

was evaluated based on annexin V-EGFP/PI and trypan blue staining immediately after thawing and

370

after culturing for 24 h (Table 3). Non-frozen thymic cells prepared by enzymatic digestion or

371

mechanical homogenization of the same thymic tissue were used as controls (viability, 97.6 ± 2.3%

372

(trypan blue staining) and 90.2 ± 1.4% (annexin V-EGFP/PI staining)). These data also indicated

373

that the most effective cryoprotective medium was CPM-7. No significant differences for frozen

374

thymic fragments in use of any selected cryoprotective media were found. However, thymic

375

fragments contained a higher percentage of dead cells (40 - 50%) for both measurements (trypan

376

blue and annexin V-EGFP/PI). This can be explained by poor penetration of the cryoprotectant into

377

thymic pieces compared to cell suspensions. Thus, we can assume that though for a total thymic cell

378

viability there is no significant difference in protective efficiency of evaluated cryoprotective

379

media, CPM7 is preferable for storage of both the cell suspensions and thymic fragments, and

380

furthermore storage of cell suspensions is preferable to storage of thymic fragments.

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381

Table 3

383

Effect of selected cryoprotective media composition and samples preparation method on cell

384

viability in frozen-thawed thymic samples evaluated by staining with trypan blue and annexin V-

385

GFP/PI.

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Cell viability with

Cell viability with

annexin V-EGFP/PI (%)

trypan blue (%)

Cryoprotective media

†

CPM1

Non-cultured †Cultured 49.4 ± 3.2

60.3 ± 6.8

♀

Fragments

†

Non-cultured

†

Cultured

97.2 ± 2.3

♀

Fragments

45.7 ± 5.7

87.2 ± 2.7

57.2 ± 5.8

CPM2

*53.7 ± 6.3 *44.8 ± 9.2 *52.9 ± 6.9

*78.9 ± 3.1

*95.6 ± 4.1 *55.4 ± 4.6

CPM7

**74.5 ± 3.8 *71.3 ± 7.6 *49.4 ± 2.4

*90.5 ± 1.9

*98.1 ± 1.4 *52.7 ± 3.2

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386

†

387

30 days and analyzed by flow cytometry (annexin V-EGFP/PI staining) and light microscopy

388

(trypan blue staining) for cell viability directly after thawing (non-cultured) and after culturing for

389

24 h (cultured). ♀Thymic fragments (0.8 – 1.0 cm3) were stored in liquid nitrogen for 30 days, and

390

cell suspensions were prepared by mechanical homogenization and analyzed for cell viability as

391

above. Data show mean ± SD for n = 3 (three individual thymic samples for each CPM from the

392

same thymus were analyzed). *p > 0.05 and **p < 0.01 compared to samples frozen in CPM1.

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Thymic samples were prepared by enzymatic digestion, and cells were stored in liquid nitrogen for

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3.2. Effect of cryprotective media compositions and samples preparation method on viability of

395

CD326+ thymic epithelial cells

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To specifically determine the level of CD326+ TEC viability in frozen thymic cell suspensions

398

prepared by enzymatic digestion, staining with PI and anti-CD326 Abs was performed after thawing

399

of the samples. The data indicate a high level of CD326+ cell survival (> 90%) in three selected

400

CPM (Fig. 2). At the same time, the overall percentage of CD326+ cells among thymic cells was

401

only about 10 - 15%. These data show that CPM7 was also comparatively effective for protection of

402

CD326+ TEC in frozen thymic samples.

403

Fig. 2.

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Analogous experiments were carried out with wide set of CPM for enzymatically digested

405

thymic tissues as well as for thymic fragments (Table 4) where a commercial cryoprotective

406

medium SCB was also used for comparison, and cell suspensions were prepared from several

407

individual thymuses. In these experiments, cell samples were stained with 7-AAD for cell viability

408

evaluation and with anti-CD326, to evaluate the percentage of viable TEC in the cell samples.

409

Although there was no significant difference in the cryoprotective efficiency of the tested CPM on

410

total thymic cell suspension, the overall conclusion was that CPM7 was preferable for storage of

411

both thymic samples types. The enzymatic method of thymic samples preparation intended for

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storage was more effective then storage of thymic fragments as evaluated by total cell viability.

413

However, it was not clear tendency in terms of CD326+ epithelial cells.

414

Table 4

416

Effect of cryoprotective media composition and samples preparation method on cell viability of

417

CD326+ epithelial cells in frozen-thawed thymic samples stained by 7-AAD.

media

Cell suspensions

Viable cells (%)

CD326+ cells (%)

†

Thymic fragments

Viable cells (%)

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♀

Cryoprotective

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CD326+ cells (%)

83.03 ± 4.15

4.08 ± 0.78

CPM2

*75.89 ± 9.83

1.22 ± 0.40

CPM3

91.16 ± 2.15

CPM4

86.48 ± 0.65

CPM5

81.91 ± 3.46

CPM6

69.68 ± 9.27

CPM7

84.08 ± 2.40

*7.66 ± 1.21

62.37 ± 9.13

4.95 ± 0.76

Not tested

Not tested

*#1.84 ± 0.23

*81.83 ± 3.68

*6.61 ± 1.58

56.33 ± 9.73

2.62 ± 0.57

2.92 ± 0.75

Not tested

Not tested

6.42 ± 0.62

Not tested

Not tested

4.17 ± 0.72

Not tested

Not tested

5.55 ± 0.88

*50.23 ± 0.7

*1.18 ± 0.15

*75.73 ± 4.23

*9.36 ± 1.37

86.43 ± 3.90

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2.78 ± 0.32

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CPM8

65.78 ± 3.15

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♀

419

nine CPM compositions and stored in liquid nitrogen for 30 days. Three random cell samples for

420

each thymus were evaluated after thawing by flow cytometry. †Thymic fragments were frozen in

421

five CPM compositions and stored in liquid nitrogen for 30 days. Three random thymic fragments

422

for each thymus were enzymatically digested after thawing, and individual cell suspensions were

423

evaluated by flow cytometry. Data show mean ± SD for five thymi; n = 15 per condition. *p < 0.05

424

compared to samples frozen in CPM1; #p < 0.05 compared to samples frozen in CPM7.

425

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Individual thymic samples were prepared by enzymatic digestion. Cell samples were frozen in

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426

3.3. Effect of storage time and samples preparation method on viability of CD326+ epithelial

427

thymic cells

428

Next, we analyzed TEC amount after different time of storage in liquid nitrogen.

430

Enzymatically digested cells were stored in liquid nitrogen up to 6 months in CPM-7. Data showed

431

that the number of TEC was decreased with increasing of storage time in liquid nitrogen (Fig. 3).

432

After 7-day storage in liquid nitrogen, the number of CD326+ cells was decreased three-fold

433

compared to freezing and thawing, and was relatively stable until day 30. However, it subsequently

434

dropped a further ten-fold by day 180.

435

Fig. 3.

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To evaluate the efficiency of CD326+ epithelial cell survival in stored cell suspensions compared

437

to thymic fragments, cell suspensions and thymic fragments were prepared from three individual

438

thymuses, frozen in CPM7 and analyzed on days 1, 7, 14 and 30 (Fig. 4). Both types of frozen

439

thymic samples showed a two-fold decrease in CD326+ epithelial cells in stored thymic samples by

440

day 30, with similar dynamics. However, the percentage of viable TEC was higher in frozen thymic

441

fragments.

442

Fig. 4.

443

3.4. Effect of cryprotective media compositions and samples preparation method on viability of

444

different thymic cell types

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Finaly we analyzed the effect of CPM1 and CPM7 on cell viability for different thymic cell types,

447

in comparison to the commercial cryoprotective medium SCB (Fig. 5). Here, cell samples prepared

448

by enzymatic digestion before freezing, and frozen thymic fragments with enzymatic digestion after

449

thawing were analyzed. After analysis of frozen cell suspensions, CPM7 showed equivalent

450

efficiency to CPM-1 and SCB for CD45+ common lymphoid population and FSP+ fibroblasts but

451

was more effective for CD326+ TEC and STRO-1+ mesenchymal cells (Fig. 5 A). Similar results

20

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452

were obtained for frozen thymic fragments, except for CD45+ common lymphoid population, which

453

in case of CPM1 was less effective (Fig. 5 B).

454

Fig. 5.

455

3.5. Effect of cryprotective media compositions and samples preparation method on monolayer

457

formation by thymic stromal-epithelial cells

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458

Effect of different cryprotective media compositions and samples preparation method on

460

monolayer formation by thymic stromal-epithelial cells was evaluated in 12-day cultures of thymic

461

cell samples prepared by enzymatic digestion before freezing, of thymic cell samples prepared by

462

mechanical homogenization after thawing of stored thymic fragments and of small explants

463

generated from stored thymic fragments after thawing (Fig. 6). Though some of analyzed samples

464

contained a high percentage of dead cells (about 50 - 40%) after freezing-thawing procedure, all the

465

analyzed samples seeded in culture showed high ability to produce monolayer of stromal-epithelial

466

cells, with prevailing growth of fibroblast-like cells. All thymic samples that were stored in CPM-7

467

showed the best growth of stromal-epithelial cells (Fig. 6 C, F, I), and this was showing a direct

468

relationship with the effect of different CPM on the cell viability.

469

Fig. 6.

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Additionally, the efficiency of CPM7 was compared to SCB through monolayer formation by

471

enzymatically digested cell suspensions stored in liquid nitrogen for 14 days (Fig. 7) as well as cell

472

suspensions prepared by enzymatic digestion from frozen thymic fragments. These data also

473

indicated higher efficiency of CPM7 for storage of thymic cell suspensions. The efficiency was

474

evaluated on day 7 and day 14 of monolayer formation (Fig. 7 A, B). Only rare single cells were

475

found in cell cultures prepared by enzymatic digestion from thymic fragments frozen in CPM7 and

476

SCB with some growth predominance in CPM7 (data not shown).

477

Fig. 7.

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ACCEPTED MANUSCRIPT 478 479 480 481

483

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4. Discussion

484

We have tested nine different freezing media and three different sample preparation methods

486

(thymic fragments, cells suspensions obtained by enzymatic digestion and cells suspensions

487

obtained by mechanical homogenization) in order to identify an optimal freezing protocol for

488

human thymic samples. From this work, we conclude that, of these media, CPM7, which contains

489

95% dextran-40 (non-penetrating compound) plus 5% Me2SO (penetrating compound), is the best

490

overall in terms of the viability and functionality (monolayer formation and long-term growth

491

abilities) of frozen thymic fragments or cell suspensions as well as for potential clinical application

492

of the stored thymic samples. In terms of the same tested parameters, there was no significant

493

difference for CPM compositions that use the penetrating compound Me2SO in combination with

494

FBS (CPM1 and CPM2) compared to compositions that included non-penetrating compounds such

495

as hydroxyethyl starch (CPM6) and dextran-40 (CPM7) in combination with Me2SO as well as in

496

the case of single used dextran-40 (CPM8). However, some CPM compositions such as CPM3

497

(Me2SO + sucrose in DPBS), CPM4 (glycerol + RPMI-1640) and CPM5 (glycerol + 0,1 M sucrose

498

in RPMI-1640) that included the penetrating compound glycerol instead of Me2SO or low

499

concentration of Me2SO (CPM3) showed worse cryoprotective properties for cell viability but still

500

were suitable for stromal-epithelial cell growth (data not shown). This correlates with data of other

501

authors for mesenchymal cell cryoprotection, described by Marquez-Curtis et al [21], where CPM

502

with Me2SO concentration in range of 5 – 10% was considered optimal and lower concentrations

503

were not effective. Moreover, Me2SO was more effective than glycerol and other cryoprotectants

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22

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[21]. As established in our multiple analyses of thymic samples stored in liquid nitrogen from

505

several days up to 1.5 years, the percentage of viable thymic cells right after thawing should be at

506

least 20 - 25% to form a monolayer of stromal-epithelial cells successfully. If it shows less than 10 -

507

15%, the growth was very poor or was unsuccessful at the concentration of viable thymic cells less

508

than 5%, and it did not depend on the CPM used (data not shown). Taking into account possible

509

clinical use of stored thymic samples, the serum-free cryoprotective compositions such as CPM6,

510

CPM7 and CPM8 are preferable. Of these, CPM7 showed the best cryoprotective efficacy even

511

compared to the commercial GMP manufactured Stem-CellBanker cryomedium. Although, we did

512

not find any comparative investigations of different CPM compositions effect on thymic cell

513

viability and cell growth efficiency, many authors observed decreased cell viability for other tissues

514

during long-term storage in liquid nitrogen. It could lead to some loss of cell proliferation and hence

515

increased monolayer formation time by adherent cells. However, it was not critical for the cell

516

growth or cell function compared to non-frozen samples [2, 7, 13, 33, 37, 38]. In general, this

517

corresponds to our observations with thymic samples, but we can also conclude that freshly

518

prepared thymic cell suspensions from non-frozen thymic tissue form a stromal-epithelial

519

monolayer in a shorter time under the same conditions.

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504

In addition, we showed that freezing of thymic fragments (0.8 - 1 cm3 in size) can also be

521

successfully used for long-term thymic sample storage. However, this approach was less effective

522

for stromal-epithelial growth in culture, especially if thymic fragments were enzymatically digested

523

after thawing. This supports the data of Badowski et al [2] for umbilical cord tissue, who noted a

524

significant decrease of mesenchymal cell growth, if cell suspensions were received by enzymatic

525

digestion of primarily frozen tissues [2]. It may be explained by damage during enzymatic treatment

526

following freezing-thawing procedure.

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527

As our results show, the percentage of CD326+ epithelial cells in non-frozen general thymic cell

528

population was 1.5 - 10 % depending on the method of thymic cell preparation and analysis

529

protocol. CD326+ cells had a high viability rate after freezing-thawing procedure, but survived

23

ACCEPTED MANUSCRIPT

much worse during the long-term storage, especially if this exceeded 30 days. A significant

531

decrease in CD326+ cell viability was observed also in a period up to 7-day storage that may be

532

explained by necrosis of frozen cells, which were damaged by the freezing procedure. It is not in

533

line with some other observations for cord blood and adipose tissue mesenchymal cells, where long-

534

term storage did not significantly decrease cell viability [21]. Despite progressive loss of CD326+

535

epithelial cells during long-term storage, the remaining cells maintained functional activity and

536

could be successfully recovered after freezing even after 6-month storage in liquid nitrogen (data

537

not shown). In general, growth activity depends on concentration of viable cells that can be seeded

538

in primary culture. It correlates with some data regarding successful use of frozen thymic fragments

539

for immune system recovery [6, 18] as well as with functional activity data for other tissues [2, 11,

540

21].

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When we analyzed an effect of three different CPM compositions, CPM1, CPM7 and SCB, on

542

cell viability of frozen-thawed thymic samples and their cell compositions that included CD326+

543

TEC, CD45+ common lymphoid population, FSP+ fibroblasts, STRO-1+ mesenchymal cells, we did

544

not find here any significant difference in cell viability. However, CPM7 was more promising at

545

least for CD326+ TEC. Moreover, stromal-epithelial cells stored in CPM7 showed more intensive

546

growth in culture compared to other cryoprotective media.

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Summarizing these data, we can conclude that although there was no critical difference in

548

efficiency of tested cryoprotective compositions, CPM7, that did not include FBS and consisted of

549

penetrating (Me2SO) and non-penetrating (dextran-40) components, is the most suitable for long-

550

term storage of both the thymic suspensions and thymic fragments according to CD326+ TEC

551

viability and stromal-epithelial monolayer formation as well as for possible clinical application of

552

stored biomaterial.

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553 554 555

Conflict of interest

24

ACCEPTED MANUSCRIPT 556

None.

557 558

Statement of funding

559

This work has received funding from the European Union’s Seventh Programme for research,

561

technological development and demonstration, within the THYMISTEM project under grant

562

agreement No [602587].

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Acknowledgments

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We gratefully acknowledge Prof. Clare Blackburn from the MRC Centre for Regenerative

567

Medicine, University of the Edinburgh (Edinburgh, UK) for careful reading of the manuscript and

568

very value corrections and advises, Olga Karpachova and Valerii Hlinka from Research Centre of

569

Immunology and Biomedical Technologies, University “Ukraine” for technical assistance.

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Fig. 1. Visualization of necrotic and viable thymic cells in suspensions of frozen-thawed thymic

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samples stored in liquid nitrogen for 30 days in CPM1. (A) staining with annexin V-EGFP; (В) staining with ethidium bromide. (С) merge; (D) transient light. The image shows the same field in A, B, C and D variants of microscopy. The results for one representative experiment are presented.

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Arrowheads: dotted line - necrotic cells; solid line - viable cells. x320 magnification.

Fig. 2. Comparative evaluation of CD326+ cell viability in frozen-thawed thymic cell suspensions

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stored in three different cryoprotective media. Thymic tissue was prepared by enzymatic digestion, and cell suspensions were stored in liquid nitrogen for 30 days. After thawing, cell samples were stained with anti-CD326 Abs or PI, and analyzed by flow cytometry. Numbers above columns show percentage of CD326+ cells in cell samples. Numbers inside columns show percentage of viable

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CD326+ cells in the cell samples. Data show mean ± SD for n = 3 (three individual cell samples for each CPM from the same thymus showed in Table 3). p > 0.05 compared to samples frozen in

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CPM1.

Fig. 3. Survival dynamics of CD326+ epithelial cells stored in liquid nitrogen with CPM7.

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Thymic tissue was prepared by enzymatic digestion, and individual cell suspensions were stored in liquid nitrogen. Three cell samples were thawed on days 1, 7, 14, 30 and 180 after freezing. Individual cell samples were stained with anti-CD326 and 7-AAD, and analyzed by flow cytometry. Numbers in the quadrants and up the columns show the percentage of CD326+ viable cells in total One of three equivalent representative data sets is shown. FMO –

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thymic cell suspensions.

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fluorescence minus one.

Fig. 4. Survival dynamics of CD326+ epithelial cells stored in liquid nitrogen with CPM7.

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(A) Thymic tissues were prepared by enzymatic digestion, and individual cell suspensions were stored in liquid nitrogen. (B) Thymic fragments were stored in liquid nitrogen, and three random thymic samples for each thymus were prepared by enzymatic digestion after thawing. Three individual cell samples for each thymus were stained with anti-CD326 and 7-AAD and analyzed by

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flow cytometry on days 1, 7, 14, 30 after freezing. Numbers above columns show a percentage of CD-326+ viable cells in total thymic cell suspensions. Data show mean ± SD for three thymuses; n = 9 per condition. *p < 0.05 compared to non-frozen thymic cell suspensions prepared by

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enzymatic digestion (day 0).

Fig. 5. Cell composition of thymic samples after storage in liquid nitrogen for 30 days in various

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cryoprotective media. (A) Thymic tissues were prepared by enzymatic digestion, and individual cell suspensions were stored in liquid nitrogen. (B) Thymic fragments were stored in liquid nitrogen, and three random thymic samples for each thymus were prepared by enzymatic digestion after thawing. Three individual cell samples for each thymus were stained with the markers shown and

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with 7-AAD and analyzed by flow cytometry on day 30 after freezing. TEC (thymic epithelial cells) - CD326+ cells; thymocytes – CD45+ cells; fibroblasts – FSP+ cells (fibroblast surface protein); MC (mesenchymal cells) – STRO-1+ cells. Data show mean ± SD for three thymuses; n = 15 per

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condition for each CPM. *p < 0.05 compared to samples frozen in CPM1; #p < 0.05 compared to

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samples frozen in CPM7; +p < 0.05 compared to non-frozen samples.

Fig. 6. Stromal-epithelial cell monolayer formation by thymic samples frozen in three different

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cryoprotective media. Data from the same thymi that were used to obtain the data in Tables 3 and 4, are shown. (A, B, C) Cells were prepared by enzymatic digestion, stored in liquid nitrogen for 3 days and seeded into small Petri dishes (d = 3 cm) with primary concentration 2 × 107 cells/ml in 2.5 ml of culture medium CM-1. (D, E, F) Thymic fragments (0.8 – 1.0 cm) were stored in liquid

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nitrogen for 3 days, cell suspensions were prepared by mechanical homogenization after thawing, and cells were cultured as described above. (G, H, I) Thymic fragments after thawing were cut into small explants (1 - 2 mm), and 5 - 6 explants were seeded into small Petri dishes and cultured as

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described above. (G) The dark area on the image (left low corner) shows explant edge. The cell growth was evaluated using the confluency value on day 12. (А, D, G) CPM1, confluency

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approximately 60%, 50% and 55%, respectively; (В, E, H) CPM2, confluency approximately 30%, 20% and 40%, respectively; (C, F, I) CPM7, confluency approximately 80%, 60% and 65%,

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respectively. x100 magnification.

Fig. 7. Stromal-epithelial cell monolayer formation by enzymatically digested thymic cell

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suspensions frozen in CPM7 and SBC and stored in liquid nitrogen for 14 days. Cells were frozen in (A, B) CPM7 and (C, D) SCB, and cell growth was evaluated by confluency value on (A, C) day

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7 and (B, D) day 14; confluency 40%, 10% and 80%, 20%, respectively. x100 magnification.

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Highlights:

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1. Effect of different cryoprotectants on viability of human thymic samples. 2. Optimal cryoprotectant for long-term storage of thymic tissue and epithelial cells.

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3. Monolayer formation by frozen-thawed thymic stromal-epithelial cells.