Elsevier

Cryobiology

Volume 70, Issue 1, February 2015, Pages 1-8
Cryobiology

Review
Morphology, cell viability, karyotype, expression of surface markers and plasticity of three human primary cell line cultures before and after the cryostorage in LN2 and GN2

https://doi.org/10.1016/j.cryobiol.2014.10.011Get rights and content

Abstract

Primary cell line cultures from human skin biopsies, adipose tissue and tumor tissue are valuable samples for research and therapy. In this regard, their derivation, culture, storage, transport and thawing are important steps to be studied. Towards this end, we wanted to establish the derivation, and identify the culture characteristics and the loss of viability of three human primary cell line cultures (human adult dermal fibroblasts (hADFs), human adult mesenchymal stem cells (hMSCs), and primary culture of tumor cells from lung adenocarcinoma (PCTCLA)). Compared to fresh hADFs, hMSCs and PCTCLA, thawed cells stored in a cryogenic Dewar tanks with liquid nitrogen (LN2), displayed 98.20% ± 0.99, 95.40% ± 1.41 and 93.31% ± 3.83 of cell viability, respectively. Thawed cells stored in a Dry Vapor Shipper container with gas phase (GN2), for 20 days, in addition displayed 4.61% ± 2.78, 3.70% ± 4.09 and 9.13% ± 3.51 of average loss of cells viability, respectively, showing strong correlation between the loss of viability in hADFs and the number of post-freezing days in the Dry Vapor Shipper. No significant changes in morphological characteristics or in the expression of surface markers (being hADFs, hMSCs and PCTCLA characterized by positive markers CD73+; CD90+; CD105+; and negative markers CD14−; CD20−; CD34−; and CD45−; n = 2) were found. Chromosome abnormalities in the karyotype were not found. In addition, under the right conditions hMSCs were differentiated into adipogenic, osteogenic and chondrogenic lineages in vitro. In this paper, we have shown the characteristics of three human primary cell line cultures when they are stored in LN2 and GN2.

Introduction

The Andalusian Public Health System Biobank (BBSSPA) is a platform for the provision of Bioresources (samples + information) and services in the clinical and biological format. It is a Biobank network operation that covers all blood and tissues banks dedicated to the collection, processing, storage and assignment of human biological samples in the south of Spain. Thus, the fundamental role is to provide the necessary biological material to carry out the research projects related to science, at the same time as knowledge, development and derivation of new cell lines. These cell lines have very different origins and nature, and have high economic and intellectual value, so far in the BBSSPA the transportation and distribution of these cell lines between laboratories, researchers and Biobank, is a strategic process to be successfully validated to ensure the highest quality standards.

Human adult dermal fibroblasts (hADFs), human adult mesenchymal stem cells (hMSCs) and primary culture of tumor cells from lung adenocarcinoma (PCTCLA) are three human primary cell line cultures, derived and expanded from different tissue types in the BBSSPA, all of them with different characteristics, pluripotency levels and cell survival. hADFs are somatic cells that live within the dermis layer of skin which are responsible for generating connective tissue. They are derived from mesenchymal stem cells, sharing many specific properties and markers [15]. Recent studies displayed the ability to (trans) differentiate along the mesodermal and endodermal lineages [16], [20], so they do not appear to be fully differentiated [32]. These cells have an enormous plasticity that has become the center of attention after the discovery of induced cell pluripotency [37], [38].

hMSCs are adult stem cells with immunomodulatory properties, low levels of immunogenicity and great capacity of differentiation into other lineages such as osteocytes, chondrocytes, adipocytes, cardiomyocytes, and neuronal ones [18], [23], [28], [39], investigated in pre-clinical approaches for tissue regeneration [2], [7], [10], [26], with a high proliferative capacity and capable of self-renewal. hMSCs can be isolated from many different tissues (bone marrow, umbilical cord, adipose tissue, etc.), and have been suggested as a potential cell source for tissue engineering, cell-based therapy [6], [9], [19], [27] and production of conditioned medium for stem cell research [25], [29], [31], [41]. They are able to suppress immune responses, primarily suppressing T-cell proliferation and cytokine production [6], [9], [19], [36], being useful in the treatment of graft-versus-host disease [36].

Lung cancer is the most common malignant disease worldwide, and the major cause of death from cancer, with a poor prognosis with 5-year survival rates of approximately 15%. Lung cancer almost exclusively involves carcinomas, arising from epithelial tissue of the trachea, bronchi or lung [34]. The primary culture of tumor cells is expected to be useful for investigating cancer biology and predicting chemosensitivity for individual patients. However, it has been hampered by technical difficulties, including poor cell viability, weak growth, and contamination by host cells, especially fibroblasts [12].

Cryopreservation is a freezing method that allows preserving the viability of cells, without causing additional damage by increased osmotic stresses, dehydration, toxic levels of electrolytes, and the formation of intracellular ice crystals [11], [21], [22], [24]. To prevent losing temperature during transport, we used Dry Vapor Shipper containers designed for the safe transportation of biological samples at cryogenic (−196 °C) temperature. This container uses LN2, but works with GN2, and the storage temperature inside the shipping cavity remains a stable temperature at approximately −190 °C for a 10+ day dynamic shipment or until the liquid nitrogen evaporates from the absorbent material.

Thus, the aim of the present study was to determine the in vitro cell culture characteristics and loss of viability of hADFs, hMSCs and PCTCLA before and after the cryopreservation and storage in LN2 and GN2. The results showed that cell culturing under the experimental conditions described for hADFs, hMSCs and PCTCLA are not associated with morphological differences, changes in the expression of surface markers, changes in the differentiation in hMSCs and the occurrence of chromosomal aberrations. However, in function of both the elapsed time and the type of cryopreserved cell line, there is a loss of cell viability after the storage in GN2.

Section snippets

Culture of human cell lines

We analyzed three types of human cell lines widely derived, used and transported in the BBSSPA, adult dermal fibroblasts (hADFs), adult mesenchymal stem cells (hMSCs) and primary culture of tumor cells derived from lung adenocarcinoma (PCTCLA), after receiving the signed informed consent form from patients and donors. The research was carried out according to The Code of Ethics of the World Medical Association Declaration of Helsinki, and all protocols were approved by the Ethical Committee of

Morphology and cell viability after the cryopreservation, storage in LN2 and GN2, and thawing

Before the cryopreservation, the different cell lines showed a high capacity for expansion in vitro and exhibited a robust morphology (Fig. 1), according to the particular morphology of each cell line. Upon reporting, adherent viable cells were observed from all 15 vials of frozen cells. Based on increasing confluency over one week in culture, all vials produced cells with proliferative capacity, maintaining exactly the same morphology. hADFs were cells with bipolar or multipolar morphology,

Discussion

In the present work, we have checked the morphology, the expression of surface markers, the chromosomal stability, the plasticity and the viability loss of three human primary cell line cultures, each one with different origins and characteristics between them, when they are stored first in LN2 and then in a Dry Vapor Shipper with GN2, filled with liquid nitrogen weekly, for a maximum period of 20 days.

Cell morphologies were monitored at an early passage after cell isolation. Subsequently, fresh

Acknowledgments

We thank the staff at the Andalusian Public Health System Biobank (BBSSPA) for collecting the normal foreskin, adipose tissue, and lung adenocarcinoma. And, especially to the culture area that conducted the experimental work. All authors contributed to manuscript review and approval.

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    Statement of funding: This work was supported by a grant from ISCIII-CSJA, Spain: EMER07/054 to R.D.Q.

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