Back to Top

Ask about this
Protocol

Protocol

Skin Freezing Technique for Living Cell Bank

  • José Mauricio Barbanti Duarte 1,2
  • Joao Airton Boer 1,2
  • Eluzai Dinai Pinto Sandoval 1,2
  • Agda Maria Bernegossi 1,2
  • Iara Maluf Tomazella 1,2
  • 1 - Núcleo de Pesquisa e Conservação de Cervídeos (NUPECCE)
  • 2 - Faculdade de Ciências Agrárias e Veterinárias da Universidade Estadual Paulista (UNESP)

Jul 22, 2021

Abstract

Cell banks are especially important sources of studies for which living cells are needed, as well as a repository of genetic diversity in natural populations. We propose a simple protocol for field research that can maintain viable skin cells for in vitro replication and production of fibroblast lines. We use a culture medium solution prepared with antibiotics, fungicide, and external (PVP) and internal (DMSO) cryoprotectants. After equilibrating for 3 hours at 4oC, the tubes with the skin fragments are exposed to liquid nitrogen vapor for 30 min, being subsequently immersed and kept in this environment for an indefinite period.

Introduction

The use of living cells in cytogenetic research was originally important for the description of many species of animals (Hsu, 2012). More recently, fibroblasts have been used as a source of material for cloning (Campbell et al., 1996) or pluripotent cell lines such as IPSCs (Takahashi et al., 2007). These perspectives demonstrate the need for developing a simple technique to be implemented in the field, which keeps cells alive for an indefinite time of storage. Within this context, the current proposal arises from a demand to collect deer material for cytogenetic studies. The given procedural steps were based on the technique of leukocyte freezing described in Duarte et al. 1999. We adapted the duration of each step of the slow freezing procedure, the amount of culture medium and included a skin transport medium as a pre-treatment to avoid contamination. Thereby, our protocol has been used since 1994 with good results in obtaining cell cultures of deer, allowing the development of several studies in this taxonomic group (Duarte and Jorge, 2003; Abril and Duarte, 2008; Abril et al., 2010; Valeri et al., 2018; Cifuentes-Rincón et al., 2020). The success in the storage of live tissue for more than 1000 specimens of Neotropical Cervids at the Deer Research and Conservation Center (NUPECCE) cell bank reveals the necessity of sharing with the scientific community, a simple, inexpensive, and viable technique to be carried out in the field. The described technique allows the stocking of high-quality mammalian genetic material. It can be used not only in anesthetized animals for routine procedures in captive wildlife centers but also in hunted, road-killed, or killed animals for other circumstances. We believe that the maintenance of cell banks in liquid nitrogen is an inexpensive and viable way to store genetic material as a source for research or as repository of genetic diversity of wildlife populations in the near future. Therefore, it is urgent that population of animals that still demonstrate good genetic diversity have their genetic material preserved in order to maintain their evolutionary potential. The biggest advantage of our method is that it allows more than one skin fragment of a specific specimen to be kept stored indefinitely. Certainly, this technique would serve as a basis for the implementation of these banks.

Reagents and Equipment

Sample collection Materials:  

Razorblade. Gauze. Tweezers, Scissors. Triclosan antisepsis soap (Soapex 1%). 70% ethanol. 

1. Skin Divulsion at Laboratory 5 units of 2ml cryovial with skin freezing medium, 25ml Petri dish, Scalpel blade. Tips of 1mL. Micropipette 1mL. Culture medium. PBS solution. Laboratory lighter. 

2. Skin Divulsion in the field 5 units of 2ml cryovial with skin freezing medium. Tweezers, Scissors. Laboratory lighter 

3. Slow skin freezing procedure Fridge, Styrofoam box, Cryovial support, Ring stand, Rachis liquid nitrogen canister. 

Reagents:

1. PBS: Add NaCl 10g in 1L of ultra-pure water. Proceed adding KCl 0.25g, Na2PO4 + 12H2O 1.44g and finally KH2PO4 0.25g. Remain under agitation for 15 minutes using a magnetic agitator. Adjust the pH to 7.2. Filter through a 0.22um membrane. Freeze. 

2. Culture medium: Add 10,15 g/ 1000mL DMEM medium (Meio Dulbecco MEM. Cat. Cultilab n°4706) lyophilized in 1L of ultra-pure water Add 3.7g of sodium bicarbonate. Add 1% of Penicillin-Streptomycin solution 10.000ug/mL (lot. Hyclone n° 094341) Add 2mg/mL of Amphotericin B 1mg/L (lot. Cultilab n° 4707). Adjust the pH to 6.8 - 7.2. Remain under agitation for 15 min. Filter through a 0.22um membrane. Store in a refrigerator (4 ° C) for up to two months. At the time of use, add 10% FBS. 

3. Skin transport medium: Culture medium supplemented with 9% of Penicillin-Streptomycin solution 10.000ug/mL (lot. Hyclone n° 094341) and 10mg/mL of Amphotericin B 1mg/L (lot. Hyclone n° 094341). 

4. Skin freezing medium: 50 ml de Culture medium. Add 100mg/ml de Polivinil (12g/100mL). Remain under agitation. Add another 50 mL de culture medium and 20 ml of FSB. Add 6,25g of DMSO (lyophilized). Add 2mg/mL of Amphotericin B 1mg/L. Remain under agitation for 3h. Add 1% of Penicillin- Streptomycin solution 10.000ug/mL. Freeze until use Thaw at room temperature before using. 

Equipment:

Laminar Flow Hoods (for laboratory procedure). Fridge at 5°C. Container with liquid nitrogen (-196°C). The schematic representation of the procedure is shown in figure 4.


Procedure

The established protocol allows you to freeze mammalian skin samples from anesthetized or slaughtered animals (less than 12h of being slaughtered). To avoid contamination, collect the sample in a previously disinfected environment. Place the specimen on a sterile blanket and perform all the steps using sterile gloves. 

1. Sample Collection Procedure

  1. Select an area in the inner thigh medially between the alba line at the inguinal region (left or right). (Figure 1A)
  2. Wash the selected area with 1% Triclosan antiseptic soap (Soapex 1%), spreading out the region to properly distribute the soap solution.
  3. Shave the area where the soap was applied using a razor blade.
  4. Spray ethanol 70% in each part of the region previously shaved.
  5. Dry with sterile gauze.
  6. Repeat step 4.
  7. Dry with sterile gauze.
  8. Lift a 2x5 cm area with tweezer and cut at the base with sterile scissors. (Figure 1B)
  9. Place the fragment in a cryovial with skin transport medium at room temperature and take it to the lab. If the sample takes more than 30 min to arrive in the lab, maintain it in a stake box with ice until you arrive in the lab (max 24 h). The field laboratory can be a tent or a closed room, to avoid the wind.

Figure 1. Sample collection procedure. (A) Area selected for skin fragment collection: red square; alba line: blue long dash. (B) Skin Fragment collection.

2. Skin Fragment Divulsion

2.1 Skin Fragment Divulsion in the field

In field collections, where there is no possibility of fragmenting the sample in sterile

environment or the laboratory, divide the skin fragment next to the lighter as follow:

  1. Lift the skin fragment without completely removing from the skin transport medium using a tweezer. This material should be close to the lamp flame at a maximum distance of 10cm. Divide all fragments near the flame to avoid contamination. (Figure 2)
  2. Cut a small fragment of 1x1cm² using a sterile scissor and place it in one of the cryogenic tubes with the skin freezing medium.
  3. Repeat step 2 until you have placed all the cut fragments in each of the cryovial. Depending on the size of the initial fragment or the amount of sample required, the skin can be divided into 5 or 10 fragments.

Figure 2. Skin Fragment divulsion in the field. (A) Materials used for skin division. (B) Procedure and distribution of skin fragments in the cryovials with skin freezing medium.

2.2 Skin Fragment Divulsion at Laboratory

  1. In the laboratory, turn on the laminar cabin, lighter and prepare the materials for the division of the skin fragment (see materials and equipment).
  2. Remove the skin fragment from the transport medium with a tweezer, and place in a petri dish with 2 ml of PBS.
  3. Position the skin so that all sides come into contact with the PBS and transfer the fragment to another petri dish containing 2 ml of culture medium.
  4. Divide the skin into 5 small fragments of 1x1cm² using a scalpel blade and a tweezer.
  5. Separate 5 cryovial with skin freezing medium and place a fragment in each of the tubes

3. Slow Skin freezing procedure

  1. Identify each cryovial and refrigerate (4 ° C) for 3 hours.
  2. Prepare a Styrofoam box with liquid nitrogen. Place a cryovial support at 1 to 2 cm above the nitrogen level. (Figure 3)
  3. Place the cryotubes in the support inside the Styrofoam box for 30 min to continue the slow freezing in nitrogen vapor. Stabilize the support with a laboratory ring stand avoiding the contact of cryovials with liquid nitrogen.
  4. After 30 min in nitrogen vapor immerse the samples in liquid nitrogen.
  5. Place each tube in a rack and maintain in liquid nitrogen at -196 ° C, where it will remain stored.

Figure 3. Slow skin freezing procedure. (A) Material used for Styrofoam box adaptation. (B) Cryovial supported at 1 to 2 cm above the nitrogen level.

Figure 4. Schematical representation of the Skin Freezing Technique.

 


References

Abril, V. V., Duarte, J. M. B. (2008). Chromosome polymorphism in the Brazilian dwarf brocket deer, Mazama nana (Mammalia, Cervidae). Genet Mol Biol 31: 53–57. doi: 10.1590/S1415-47572008000100011

Abril, V. V., Carnelossi, E. A., González, S., Duarte, J. M. B. (2010). Elucidating the evolution of the red brocket deer Mazama americana Complex (Artiodactyla; Cervidae). Cytogenet Genome Res 128: 177–187. doi: 10.1159/000298819

Campbell, K. H., McWhir, J., Ritchie, W. A., Wilmut, I. (1996). Sheep cloned by nuclear transfer from a cultured cell line. Nature. 380 (6569): 64–6. doi:10.1038/380064a0

Cifuentes-Rincón, A., et al. (2020). Designation of a neotype for Mazama americana (Artiodactyla, Cervidae) reveals a cryptic new complex of brocket deer species. ZooKeys 958:143–164. doi: 10.3897/zookeys.958.50300

Duarte, J. M. B., M. F. D. Ramalho, V. F. H. Lima, and W. Jorge. (1999). A leukocyte cryopreservation technique for cytogenetic studies. Genetics and Molecular Biology 22: 399–400. doi: 10.1590/S1415-47571999000300019.

Duarte, J. M. B., Jorge, W. (2003). Morphologic and cytogenetic description of the small red brocket (Mazama bororo Duarte, 1996) in Brazil.Mammalia 67: 403–410. doi: 10.1515/mamm.2003.67.3.403

Hsu, T. C. (2012). Human and Mammalian Cytogenetics: An Historical Perspective. Springer Science & Business Media. ISBN 978-1-4612-6159-9.

Takahashi, K., Okita, K., Nakagawa, M., Yamanaka, S. (2007). Induction of pluripotent stem cells from fibroblast cultures. Nat Protoc 2 (12): 3081-9. doi: 10.1038/nprot.2007.418.

Valeri, M. P., Tomazella, I. M., Duarte, J. M. B. (2018). Intrapopulation chromosomal polymorphism in Mazama gouazoubira (Cetartiodactyla; Cervidae): The emergence of a new species? Cytogenet Genome Res 154: 147–152. doi: 10.1159/000488377

 


Comments

Be the first to comment on this protocol.

Submit a Comment

First Name *

Last Name *

Email *

Comments *

By submitting this form, you agree to the Genetic Engineering & Biotechnology News (GEN) privacy policy.

Premier Sponsor
Halo Labs