Carboxylated Poly-l-Lysine as a Macromolecular Cryoprotective Agent Enables the Development of Defined and Xeno-Free Human Sperm Cryopreservation Reagents.

Hiroki Takeuchi,Kuniaki Toriyabe,Kayo Tanaka,Suong-Hyu Hyon,Manabu Kato,Tadashi Maezawa,Yuki Gen,Eiji Kondo,Tomoaki Ikeda,Masafumi Nii,Yuko Kitano,Kento Terada-Yoshikawa,Mikiko Nishioka,Ryota Tachibana

doi:10.3390/cells10061435

Hiroki Takeuchi, Kuniaki Toriyabe + Show 12 more

Open Access

https://doi.org/10.3390/cells10061435

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Journal: Cells	Publication Date: Jun 8, 2021
Citations: 4	License type: CC BY 4.0

Affiliation: Mie University Hospital, Mie University, BMG (Japan)

Abstract

In human sperm cryopreservation, test yolk buffer and human serum albumin have been used as permeating macromolecular-weight cryoprotectants. In clinical reproductive medicine, human serum albumin is frequently used because of low risks of zoonoses and allergic reactions. However, the risk of allogeneic infectious diseases exists, and the supply may be unstable because human serum albumin is derived from human blood. Therefore, the development of xeno-free human sperm cryopreservative reagents that could overcome the aforementioned problems is warranted. We succeeded in developing a new xeno-free and defined sperm cryopreservation reagent containing glycerol, carboxylated poly-l-lysine, and raffinose. The cryopreservation reagent was not significantly different in terms of sperm motility, viability, and DNA fragmentation and was comparable in performance to a commercial cryopreservation reagent containing human serum albumin. Moreover, the addition of saccharides was essential for its long-term storage. These results may help elucidate the unknown function of macromolecular-weight permeating cryoprotective agents.

Highlights

Spermatozoal cryopreservation technology (SCPT) provides the advantage of storing sperms semi-permanently for future use [1]
Non-permeating macro-Mw cryoprotective agents (CPAs), such as test yolk buffer (TYB) [18,19] and human serum albumin (HSA) [20], should be added to the cryopreservation medium to prevent intracellular ice crystal formation induced by extracellular ice crystal formation
Sperm motility tended to be the highest in the samples treated with 5% w/v carboxylated poly-L-lysine (CPLL); we examined 5% w/v CPLL and low-Mw CPAs in detail

Summary

Introduction

Spermatozoal cryopreservation technology (SCPT) provides the advantage of storing sperms semi-permanently for future use [1] This technology is widely used to preserve genetic resources, such as those of endangered, rare [2], and laboratory animals [3,4], as well as for the efficient production of domestic animals [5] and applications associated with assisted reproductive technology (ART) [6]. Low-molecular-weight (Mw) permeating cryoprotective agents (CPAs), including glycerol (Glyc), dimethyl sulfoxide (DMSO), ethylene glycol (EG), and propylene glycol (PG), can effectively suppress cytoplasmic ice crystal formation [13]. These agents are potentially cytotoxic and should be used at low concentrations [14,15]. Non-permeating macro-Mw CPAs, such as test yolk buffer (TYB) [18,19] and human serum albumin (HSA) [20], should be added to the cryopreservation medium to prevent intracellular ice crystal formation induced by extracellular ice crystal formation

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