Abstract
The cryopreservation of red blood cells (RBCs) is very important to modern medicine. Cryoprotectants (CPAs) such as dimethyl sulfoxide (DMSO), proline, trehalose, and polyvinyl alcohol (PVA) have been used in the cryopreservation of RBCs, but the results are not satisfactory. Marinomonas primoryensis antifreeze protein (MpAFP) is a Ca2+-dependent AFP derived from Antarctic bacteria, which can prevent bacteria from freezing under extremely cold conditions and may be suitable for cryopreservation of RBCs. The active region of MpAFP is located in region IV and is called MPAFP_RIV. In this paper, the gene of region IV of MpAFP is introduced into BL21 (DE3) competent cells, and MpAFP_RIV is obtained after culture, separation, and purification. The improved splat assay is proposed, and this method first proves that MpAFP_RIV has strong ice recrystallization inhibition (IRI) activity without Ca2+. The improved splat assay is easier to operate and lower the cost compared to the traditional one, and the results are consistent with the classic sucrose sandwich assay, proving that this method can accurately detect IRI activity. The feasibility of MPAFP_RIV combined with classical CPA for cryopreservation of RBCs and the methods to increase the yield of MPAFP_RIV are proposed.
Highlights
Cryopreservation refers to the method of preserving cells [1], tissues [2], and organs [3] at extremely low temperatures
More than thirty million units of blood are consumed every year in the United States, and a patient sometimes needs to transfuse as much as 57 liters of blood [13]
Transfection is a process in which foreign DNA fragments are actively or passively introduced into eukaryotic cells to obtain a new phenotype [41], and it is the key to the successful expression of proteins in bacteria
Summary
Cryopreservation refers to the method of preserving cells [1], tissues [2], and organs [3] at extremely low temperatures. Large ice crystals formed by ice recrystallization can pierce cell membranes and cause severe mechanical damage to cells [6]. A large amount of freezing of the extracellular solution can increase the osmotic pressure and cause osmotic dehydration to cells [7]. The current golden standard CPA for cryopreservation of cells is dimethyl sulfoxide (DMSO) [16]. DMSO can permeate into cells and adjust osmotic pressure, reducing the osmotic dehydration of cells during cryopreservation [17]. DMSO is unable to inhibit ice recrystallization, so it is unable to reduce the harm caused by large ice. In addition, DMSO is a toxic organic solvent that is believed to be associated with developmental disorders and apoptosis of cells [18, 19].
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