Experimental model for establishment of hypoxia in 75 cm² culture flasks

Modelo experimental para o estabelecimento de hipóxia em balões de cultura de 75 cm²

Abstracts

In Plastic Surgery, cell culture represents the perspective of studying cellular mechanisms that guide the healing process of several tissues. Some steps of the healing process depend on physical factors as the tissular partial pressure of O2. In cell culture, it is possible to submit cells to hypoxic enviroment. The present study reports an alternative method at low cost for the establishment of hypoxic environment in cell culture flasks.

Cells, cultured; Hypoxia


A cultura de células, na Cirurgia Plástica, representa uma perspective para o estudo dos mecanismos celulares que norteiam o processo cicatricial de diversos tecidos. Algumas etapas do processo de cicatrização dependem de fatores físicos como a pressão parcial de O2. Em uma cultura de células, é possível submeter células a um ambiente hipóxico. O presente estudo relata um método alternativo de baixo custo para o estabelecimento de um ambiente hipóxico em frascos de cultura de células.

Cultura de células; Hipóxia


EXPERIMENTAL MODELS

Experimental model for establishment of hypoxia in 75 cm2 culture flasks1 1 Study developed in the Laboratory of Cell Culture, Plastic Surgery Division, Federal University of São Paulo - São Paulo Medical School

Modelo experimental para o estabelecimento de hipóxia em balões de cultura de 75 cm2

Sidney Mamoru KeiraI; Lydia Masako FerreiraII; Alfredo GragnaniIII; Ivone da Silva DuarteIV; Anelisa Bittencourt CampanerV; Marcelino de Souza Durão JrVI

IMD, PhD, Plastic Surgery Division, Federal University of São Paulo - São Paulo Medical School

IIMD, PhD, Titular and Head of the Plastic Surgery Division, Federal University of São Paulo - São Paulo Medical School

IIIMD, PhD, Plastic Surgery Division, Federal University of São Paulo - São Paulo Medical School

IVMD, PhD, Plastic Surgery Division, Federal University of São Paulo - São Paulo Medical School

VPost-graduate, Federal University of São Paulo - São Paulo Medical School

VIMD, PhD, Nephrology Division, Federal University of São Paulo - São Paulo Medical School

Correspondence

ABSTRACT

In Plastic Surgery, cell culture represents the perspective of studying cellular mechanisms that guide the healing process of several tissues. Some steps of the healing process depend on physical factors as the tissular partial pressure of O2. In cell culture, it is possible to submit cells to hypoxic enviroment. The present study reports an alternative method at low cost for the establishment of hypoxic environment in cell culture flasks.

Key words: Cells, cultured. Hypoxia.

RESUMO

A cultura de células, na Cirurgia Plástica, representa uma perspective para o estudo dos mecanismos celulares que norteiam o processo cicatricial de diversos tecidos. Algumas etapas do processo de cicatrização dependem de fatores físicos como a pressão parcial de O2. Em uma cultura de células, é possível submeter células a um ambiente hipóxico. O presente estudo relata um método alternativo de baixo custo para o estabelecimento de um ambiente hipóxico em frascos de cultura de células.

Descritores: Cultura de células. Hipóxia.

Introduction

In vitro experimental models in Plastic Surgery has been useful to the study of biochemical and biophysical aspects of cellular dynamics from different tissues manipulated in this specialty.

In skin cell culture, it is possible to standardize factors that may have influence cutaneous cells. This fact enables the study of several physical factors (temperature, characteristics of culture medium and partial pressure of oxygen1) or chemical factors (drugs2, hormones, citokynes1,3,4,5 and vitamins).

In previous literature, the establishment of hypoxic environment in culture flasks was mentioned by IANNONNE et al.6, in a study concerning free radical biosynthesis in the metabolism of organic hydro-peroxides by normal human keratinocytes.

Proposition

Hypoxic environment may be established by using incubators with simultaneous flow of air, CO2 and N2. Nevertheless, this method has in its expensive cost its main disadvantage for it is necessary to settle an incubator for this exclusive purpose.

The present study proposes an alternative method for the establishment of hypoxic environment in 75 cm2 culture flasks, with its reproducibility determined by standardization of culture medium volume, composition of hypoxic gas mixture and gas flow rate.

Method description

The 75 cm2 culture flasks with 7,0 ml of culture medium were closed with silicon stoppers (30x24x32mm) and sealed with a double layer cellulose film (Parafilm M®). Two needles (40x8 mm) were introduced in the flasks through the stopper. One of them was connected in a infusion system with the hypoxic gas mixture. The second needle was used as an output way to the gas flow (FIGURES 1, 2).

The hypoxic gas flow was maintained for 30 minutes with a constant flow of 100 ml/min and under constant agitation in a vortex shaker. All these procedures were done inside a laminar flow.

Both needles were taken away together at the end of the hypoxia session and the culture flasks were maintained sealed at 37 °C for more 30 minutes or 24 hours.

Partial pressure of oxygen in the culture medium was estabilished by gasometry in a pHmeter and gas analyser (Radiometer Copenhagën®, model ABL 330) (TABLE 1). With this hypoxia establishment procedure it was possible to lower the partial pressure of O2 to an average of 30 mmHg.

Analyses of significant differences between data sets were performed by the use of Friedman's test and Multiple Comparison test. Values of p < 0,05 were considered statistical significant.

It was observed that there is statistical significant difference (c2calc= 54,240 - p = 0,000) among the groups maintained in air, in incubation under 5% CO2 in air and after infusion of hypoxic gas mixture and maintenance of hypoxia for 30 minutes. These results show that the present method was efficient in establishing a hypoxic environment into the culture flask.

On the other, there was not statistical significant difference (TABLE 2) between the group submitted to hypoxic infusion and stabilized for 30 minutes and the group maintained sealed for 24 hours with the hypoxic environment after the infusion. This result showed that the present experimental method is effective in maintaining the hypoxic environment into the culture flask for so long as 24 hours after the hypoxic infusion procedure (FIGURE 3).

Perspectives

The present experimental model showed efficient to establish a hypoxic environment in 75 cm2 culture flasks maintained as a sealed system with the hypoxia apparatus. The theoretical basis that justifies the reproduction of this model is the standardization of culture medium volume and superficial area, duration of the hypoxic infusion and the hypoxic gas mixture gradient between the gas and liquid phase in the culture flask. So the theoretical substratum of Fick Law (theory of gas diffusion in the interface between two distinct phases) was applied to the cell culture system7.

This experimental model consists an alternative for the establishment of hypoxic environment in 75 cm2 culture flasks at low cost.

Conflito de interesse: nenhum

Fonte de financiamento: nenhum

Keira SM, Ferreira LM, Filho AG, Duarte IS, Campaner AB, Durão MS. Modelo experimental para o estabelecimento de hipóxia em balões de cultura de 75 cm2. Acta Cir Bras [serial online] 2004 Vol 19 Edição Especial. Disponível em URL: http://www.scielo.br/acb.

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  • 6. Iannonne A, Marconi A, Zambruno G, Giannetti A, Vanninni V, Tomasi A - Free radical production during metabolism of organic hydroperoxides by normal human keratinocytes. J Invest Dermatol. 1993;101(1):59-63.
  • 7. McLimans WF, Crouse EJ, Tunnah KV, Moore GE - Kinetics and gas diffusion in mammalian cell cultures systems. I: Exper Biotech Bioeng. 1968;10:725-63.

  • Correspondence to
    Sidney Mamoru Keira
    UNIFESP-EPM, Plastic Surgery Division, Surgery Division
    Rua Napoleão de Barros, 715, 4º andar
    04024-900 São Paulo - SP
    Tel: (11)557604118 FAX: (11) 55716579
  • 1
    Study developed in the Laboratory of Cell Culture, Plastic Surgery Division, Federal University of São Paulo - São Paulo Medical School

Publication Dates

  • Publication in this collection
    17 Mar 2005
  • Date of issue
    Dec 2004
Sociedade Brasileira para o Desenvolvimento da Pesquisa em Cirurgia Al. Rio Claro, 179/141, 01332-010 São Paulo SP Brazil, Tel./Fax: +55 11 3287-8814 - São Paulo - SP - Brazil
E-mail: sgolden@terra.com.br