A Novel Device Design for Sperm Cryopreservation

描述（由申请方提供）：基因工程小鼠（GEM）和大鼠（格尔）系是许多重要人类疾病的优良动物模型（考克斯，2003;绿色和哈德逊，2005）。然而，通过标准育种技术维持这些品系对设施和预算产生了不可持续的压力。因此，迫切需要开发可靠且具有成本效益的啮齿动物种质保存替代方法。与目前标准的胚胎冷冻保存相比，精子冷冻保存将是一种更简单且成本更低的方法（Thornton，1999），前提是足够数量的精子以可靠的方式在冷冻和解冻后存活。然而，冷冻保存后的小鼠精子存活率在不同品系之间是高度可变的（Mazur等人，2000; Critser和Mobraaten，2000; Yildiz等人，2007年）。对于大鼠精子，迄今为止只有一组报道了成功的、可重复的冷冻保存（Nakatsukasa等人，2001年，2003年），存活率极低（<10%）。啮齿动物精子具有不寻常的形态，并且也对机械非常敏感（Katkov和Mazur，1998; Mazur等人，1998; Nakatsukasa等人，2001; Si等人，2006; Walters等人，2005年）。与这种机械敏感性相关，小鼠精子容易受到与冷冻保存期间形成的细胞外冰的形态相关的损伤。然而，目前用于开发冷冻保存策略的范例集中在诸如细胞内冰的形成和冷冻期间高的细胞内/细胞外溶质浓度等因素，而不是细胞的机械敏感性或冰-细胞相互作用（Mazur 1977，1984; Muldrew和McGann 1994）。因此，新技术，以防止细胞外冰损伤应应用于提高啮齿动物精子冷冻保存后的存活率。因此，本提案的总体目标是建立一种啮齿动物精子冷冻保存方法，以防止与细胞外冰形成相关的细胞机械损伤。为此，将生产一种新的装置，以在冷冻保护剂溶液中产生相对大且光滑的冰晶的定向生长，并将精子尾部定向在冰生长的相同方向上。这将通过应用介电泳取向和定向凝固技术与结晶控制来实现。所提出的装置的生产将需要：（1）生产具有嵌入的珀耳帖电极的定向固化台，以精确地控制温度梯度和冰形态并保持冰生长方向;（2）生产具有冰生长通道的操作芯片，其也将包含细胞并可以装载在定向固化台上;以及（3）产生介电泳定向子系统，以在冰生长通道内提供适当的电场梯度和大小，使得精细胞可以通过介电泳力沿与冰生长相同的方向定向。通过这种方式，可以减小由冰晶施加在精子尾部上的压缩/拉伸和剪切应力，以防止它们的机械损伤。如果小鼠和大鼠解冻后精子活力达到60%，则认为该提议的新型系统达到了最终可行性。在第二阶段，该设备将进一步优化和开发，以便于最终使用和生产。公共卫生相关性：基因工程小鼠（GEM）和大鼠（格尔）系是许多重要人类疾病的非常有用的模型;然而，通过标准育种技术维持这些动物是非常困难和昂贵的。因此，迫切需要开发可靠和具有成本效益的方法来保存小鼠和大鼠的遗传信息。与目前的标准方法胚胎冷冻保存相比，精子冷冻保存将是一种更简单且成本更低的方法。该项目将优化一种设备和方法，用于有效冷冻这些有价值的研究动物的小鼠和大鼠精子。

DESCRIPTION (provided by applicant): Genetically engineered mouse (GEM) and rat (GER) lines are excellent animal models for many important human diseases (Cox, 2003; Green and Hudson, 2005). However, maintenance of these lines by standard breeding techniques is producing unsustainable pressure on facilities and budgets. Consequently, there is a critical need to develop reliable and cost-effective alternative means of rodent germplasm preservation. Sperm cryopreservation would be a simpler and substantially less costly approach compared to embryo cryopreservation which is currently standard (Thornton, 1999), provided that sufficient numbers of sperm survive freezing and thawing in a reliable manner. However, mouse sperm survival rates after cryopreservation are highly variable among different strains (Mazur et al., 2000; Critser and Mobraaten, 2000; Yildiz et al., 2007). For rat sperm, to date there is only one group that has reported successful, repeatable cryopreservation (Nakatsukasa et al., 2001, 2003) and the survival rate was extremely low (<10%). Rodent sperm have an unusual morphology and are also extremely mechanically sensitive (Katkov and Mazur, 1998; Mazur et al., 1998; Nakatsukasa et al., 2001; Si et al., 2006; Walters et al., 2005). Related to this mechanical sensitivity, mouse sperm are vulnerable to damage associated with the morphology of the extracellular ice formed during cryopreservation. However, current paradigms for developing cryopreservation strategies focus on factors such as the formation of intracellular ice and the high intra/extracellular solute concentrations during freezing, rather than the mechanical sensitivity of cells or ice-cell interactions (Mazur 1977, 1984; Muldrew and McGann 1994). Therefore, novel techniques to prevent extracellular ice damage should be applied to improve rodent sperm survival rates after cryopreservation. It is therefore the overall goal of this proposal to establish a rodent sperm cryopreservation approach to prevent the mechanical damage to cells associated with extracellular ice formation. To that end, a new device will be produced to generate a directional growth of relatively large and smooth ice crystals in the cryoprotectant solutions and orientate sperm tails in the same direction of the ice growth. This will be achieved by applying the dielectrophoretic orientation and directional solidification techniques with electrothermal controls. Production of the proposed device will entail: (1) production of a directional solidification stage with embedded Peltier electrodes to accurately control the temperature gradient and ice morphology and maintain ice growth direction; (2) Production of an operational chip with ice growth channels which will also contain cells and can be loaded on the directional solidification stage; and (3) Production of a dielectrophoretic orientation subsystem to provide a proper electric field gradient and magnitude inside the ice growth channels so that sperm cells can be orientated in the same direction as the ice growth via the dielectrophoretic force. In this way, the compressive/tensile and shearing stresses exerted by ice crystals on sperm tails can be diminished to prevent their mechanical damage. Ultimate feasibility of this proposed novel system will be considered achieved if 60% post-thaw mouse and rat sperm motility is achieved. In Phase II, the device will be further optimized and developed for ease of end use and production. PUBLIC HEALTH RELEVANCE: Genetically engineered mouse (GEM) and rat (GER) lines are very useful models for many important human diseases; however maintaining these animals by standard breeding techniques is very difficult and expensive. Consequently, there is a critical need to develop reliable and cost-effective means of preserving mouse and rat genetic information. Sperm cryopreservation would be a simpler and substantially less costly approach compared to embryo cryopreservation which is currently the standard approach. This proposed project would optimize a device and method for effective freezing of mouse and rat sperm from these valuable research animals.