**2.4 Cell poration with ATP**

18 Current Frontiers in Cryopreservation

sucrose and trehalose has been known for years, conventional cryopreservation protocols have generally not employed them even though early work with them demonstrated their ability to protect proteins and membrane vesicles during freezing (Rudolf & Crowe, 1985; Crowe et al., 1990). Trehalose has both major advantages and disadvantages for potential preservation of mammalian cells. On the negative side mammalian cells do not have an active trehalose transport system for uptake of trehalose from the extracellular environment, while on the plus side if you can get it in mammalian cells it is not metabolized giving the opportunity for trehalose to be accumulated to potentially effective preservation concentrations. The purpose of the studies presented here were; 1) to assess or review alternative strategies for delivery of trehalose into mammalian cells, and; 2) to determine whether the benefits were specific to trehalose by investigating alternative sugars

Cells used in these studies are described in Table 1. All were grown and maintained at 37oC

**Description Acronym Culture conditions** 

A10 (ATCC# CRL-1476)

CPAE (ATCC# CCL-209)

> A7R5 (ATCC# CRL-1444)

> BCE (ATCC# CRL-2048)

The pore-forming protein H5 was obtained from the lab of Hagan Bayley (Bayley, 1994). It is derived from the bacterial toxin α-hemolysin, which forms constitutively opened pores in cell membranes. The modified bacterial toxin has been engineered to form pores in the membrane that can be opened and closed by the addition of Zn+. More specific details are

DMEM\* (4.5 g/L) 10% FCS 1.0 mM sodium pyruvate

EMEM\*\* 10% FCS 1 mM sodium pyruvate 2 mM glutamine 1X non-essential amino acids

DMEM (4.5 g/L) 10% FCS 1.0 mM sodium pyruvate

DMEM (4.5 g/L) 10% FCS 1 mM sodium pyruvate 4 mM glutamine

employing the same loading strategies.

Rat aortic myofibroblast cells

Bovine calf pulmonary artery endothelial cells

Rat aortic smooth muscle cells

Bovine corneal endothelial cells

\*Dulbecco's Modified Eagle's Medium \*\*Eagle's Minimum Essential Medium

Table 1. Cell types

**2.2 Cell poration with H5** 

**2. Materials and methods** 

**2.1 Cell culture** 

in 5% carbon dioxide.

Cells were plated at 10,000-20,000 cells/well and placed in culture. The next day, the cells were washed with poration buffer (phosphate-buffered saline [PBS] with 1X essential amino acids, 1X Vitastock, 5.5 mM glucose) designed to optimize binding of ATP4- to the receptor and facilitate formation of the pore. The cells were then placed in 50 µl poration buffer, pH of 7.45, with 0.2M trehalose. A stock solution of 100 mM ATP4-, pH of 7.45, was made fresh and added to each well to achieve a final concentration of 5 mM. After addition of the ATP4-, the cells were left at 37oC for 1 hour to allow sugar uptake. Following incubation, 200 µl of DMEM plus 1 mM MgCl2 was then added to the cells at 37oC to close the pores. After 1 hour of recovery from the loading procedure cryopreservation was initiated.
