Hubel A, Norman J, Darr T B
Biomedical Engineering Center, University of Minnesota, Minneapolis, Minnesota 55455, USA.
Cryobiology. 1999 Mar;38(2):140-53. doi: 10.1006/cryo.1999.2157.
The freezing responses of hematopoietic progenitor cells isolated from normal donors and from donors with mucopolysaccharidosis type I (MPS I) were determined using cryomicroscopy and analyzed using theoretical models for water transport and intracellular ice formation. The cells from donors with MPS I used in this investigation were cultured and transduced with a retroviral vector for the alpha-l-iduronidase (IDUA) enzyme in preclinical studies for human gene therapy. The water transport and intracellular ice formation (IIF) characteristics were determined at different time points in the culture and transduction process for hematopoietic progenitor cells expressing CD34 antigen from donors with MPS I and from normal donors. There were statistically significant changes in water transport, osmotically inactive cell volume fraction, and permeability between cells from different sources (normal donors vs donors with MPSI) and different culture conditions (freshly isolated vs cultured and transduced). Specifically, Lpg and Ea increased after ex vivo culture of the cells and the changes in permeability parameters were observed after as little as 3 days in culture. Similarly, the IIF characteristics of hematopoietic progenitor cells can also be influenced by the culture and transduction process. The IIF characteristics of freshly isolated cells from donors with MPS I were statistically distinct from those of cultured and transduced cells from the same donor. The ability to cryopreserve cells which are cultured ex vivo for therapeutic purposes will require an understanding of the biophysical changes resulting from the culture conditions and the manner in which these changes influence viability.
使用低温显微镜测定从正常供体和I型黏多糖贮积症（MPS I）供体分离的造血祖细胞的冷冻反应，并使用水运输和细胞内冰形成的理论模型进行分析。本研究中使用的来自MPS I供体的细胞在人类基因治疗的临床前研究中进行培养，并用逆转录病毒载体转导α-L-艾杜糖醛酸酶（IDUA）。在来自MPS I供体和正常供体的表达CD34抗原的造血祖细胞的培养和转导过程中的不同时间点，测定水运输和细胞内冰形成（IIF）特征。不同来源（正常供体与MPSI供体）和不同培养条件（新鲜分离与培养和转导）的细胞之间，水运输、渗透惰性细胞体积分数和通透性存在统计学上的显著变化。具体而言，细胞离体培养后Lpg和Ea增加，培养仅3天后就观察到通透性参数的变化。同样，造血祖细胞的IIF特征也会受到培养和转导过程的影响。来自MPS I供体的新鲜分离细胞的IIF特征与来自同一供体的培养和转导细胞的IIF特征在统计学上有明显差异。为了治疗目的而对离体培养的细胞进行冷冻保存的能力将需要了解培养条件导致的生物物理变化以及这些变化影响细胞活力的方式。
