CN113768138A - 一种用于细胞培养肉的可食用的结冷胶/明胶3d支架的制造方法 - Google Patents
一种用于细胞培养肉的可食用的结冷胶/明胶3d支架的制造方法 Download PDFInfo
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Abstract
本发明是一种用于细胞培养肉的可食用的结冷胶/明胶3D支架的制造方法，以所述方法制造的结冷胶/明胶3D支架可用于骨骼肌细胞的培养，为细胞培养肉的制造提供支撑，属于未来食品科技领域。本发明所述方法通过将一定浓度的结冷胶和明胶按照一定比例均匀混合后，于室温下反应10 min以上，即得到结冷胶/明胶3D支架。本发明的制造方法简单高效，不经冷冻干燥即可直接用于细胞培养，在不使用有毒交联剂的情况下，通过结冷胶和明胶之间的静电相互作用形成3D支架，得到的结冷胶/明胶3D支架具有良好的细胞粘附性和相容性，可以进行骨骼肌细胞培养，并且可以在制备过程中加入其他物质以获得具有所需质构特性和细胞黏附性的不同3D支架。本发明为细胞培养肉的研究提供了一种新型、无毒的3D支架的制造方法。
Description
技术领域
本发明涉及未来食品科技领域，具体涉及一种用于细胞培养肉的可食用的结冷胶/明胶3D支架的制造方法。
背景技术
动物蛋白质具有很高的营养价值，其蛋白质的种类和结构更加接近人体的蛋白结构和数量，而且大多都含有人体必需的8种氨基酸。然而，传统的肉类生产方式由于其对环境、公共健康和动物福利的负面影响引发了极大的关注，针对这些问题，人们提出了一系列减少温室气体排放和解决传统肉类生产方式中动物福利问题的策略。其中，除了改变传统肉类生产系统之外，各种人造肉产品也有利于减轻这些负面影响。
目前，植物性肉类成为一种受欢迎的肉类产品替代品，然而研究发现植物性肉类缺乏动物性肉制品所具有的味道和其他感官属性，一些忠实的肉食者对此并不感兴趣。除此之外，营养学家普遍认为目前市售的植物肉是经过高度加工的产品，全美养牛人牛肉协会(National Cattlemen’s Beef Association)也认为真正的牛肉具有更高的营养价值。因此细胞培养肉成为对人们更具吸引力的选择。
细胞培养肉是在体外培养动物细胞得到的真正的肉。利用从动物身上提取的细胞，通过组织工程技术培养成肌肉和脂肪组织，并被构造成肉制品，从而在不屠宰动物的情况下生产可食用的肉。细胞培养肉不仅能在一定程度上解决环境问题，降低人畜共患病和其他疾病的风险，还能为肉食者提供珍贵的动物源蛋白质。
3D支架的制备是组织工程中的关键技术，其重点在于模拟天然组织的结构和成分，使细胞能够在支架内实现三维分布并大量增殖分化形成完整的肌肉组织。目前大多数3D支架中通常存在一定量的细胞外基质，如胶原蛋白、RGD肽等，才能保证良好的细胞粘附性。然而具有良好生物活性的细胞外基质通常有提取工艺复杂、成本高的缺点，不适用于细胞培养肉。因此需要一种3D支架能够使细胞大量增殖分化，并且克服常见的3D支架成本昂贵的不足。
结冷胶是一种高分子线性阴离子多糖，由假单胞杆菌发酵产生，作为增稠剂、稳定剂广泛应用于食品中，具有生产周期短、安全无毒的优良特点。结冷胶不溶于冷水，但加热即溶解成透明的溶液，冷却后，形成透明且坚实的凝胶。明胶是一种无脂肪的蛋白质，由胶原经过变性降解得到，是一种天然营养型的食品增稠剂，有增强细胞黏附性的特点，广泛应用于组织工程中。目前常见的3D支架仍然需要使用一些有毒交联剂，如戊二醛、EDC/NHS等，来增强支架的稳定性，但无法用于生产细胞培养肉。
因此，为了制备出适用于细胞培养肉的3D支架，我们发明了一种用于细胞培养肉的可食用的结冷胶/明胶3D支架的制造方法：将一定浓度的结冷胶和明胶按照一定比例均匀混合后，于室温下反应10 min以上，即得到结冷胶/明胶3D支架。本发明的制造方法简单高效，不使用有毒交联剂，不用冷冻干燥即可直接用于细胞培养，利用结冷胶和明胶之间的静电相互作用以及结冷胶冷却后即可形成凝胶的特点制备3D支架。得到的结冷胶/明胶3D支架具有良好的细胞粘附性和相容性，并且可以在制备过程中加入其他物质以获得具有所需质构特性和细胞黏附性的不同3D支架，可以进行骨骼肌细胞培养，用于生产细胞培养肉。
发明内容
本发明的目的在于针对现有细胞培养肉研发成本高、细胞无法大量增殖等不足，提供一种用于细胞培养肉的可食用的结冷胶/明胶3D支架的制造方法。
为了达到以上目的，本实验公开了一种可食用的结冷胶/明胶3D支架的制造方法，利用结冷胶和明胶之间的静电相互作用形成水凝胶，经室温反应后得到3D支架。其中，所述的结冷胶浓度为0.5~3%，所述明胶浓度为0.5~5%。
所述明胶溶液和结冷胶溶液的质量比为1:5~5:1；其中，混合溶液在加入模具时温度保持在40℃以上，室温下放置反应10 min以上，得到3D凝胶支架。
本发明的优点：
1.本发明选用结冷胶和明胶作为原料，两种原料均安全无毒，广泛应用于食品添加剂，已实现商业化。因此，选用这两种原料制造可食用的3D凝胶支架对细胞培养肉和组织工程领域的应用具有重要价值；
2.本发明的制备方式简单高效，利用结冷胶溶液在室温下为凝胶的特性以及结冷胶和明胶之间的静电相互作用，与其他交联方式相比，无任何副产物且无需加入带有毒性的交联剂，并且可以在制备过程中加入其他物质以获得具有所需质构特性和细胞黏附性的不同3D支架，在细胞培养肉和组织工程领域具有巨大的应用潜力；
3.相较于其他常见的3D支架，本发明制备的3D支架无需使用冷冻干燥即可直接用于细胞接种，大大降低了细胞培养肉的成本以及减少了能源消耗。
附图说明
图1为结冷胶/明胶3D支架的照片；
图2为结冷胶/明胶3D支架的凝胶强度，其中A、B、C、D、E、F分别代表不同比例的结冷胶和明胶；
图3为结冷胶/明胶3D支架培养C2C12小鼠成肌细胞3天后活/死细胞染色图，其中A、B分别代表不同比例的结冷胶和明胶；
图4为结冷胶/明胶3D支架培养C2C12细胞5天后的细胞骨架染色图，其中A、B分别代表不同比例的结冷胶和明胶；
图5为结冷胶/明胶3D支架培养小鸡骨骼肌卫星细胞5天后的细胞骨架染色图，其中A、B分别代表不同比例的结冷胶和明胶；
图6为结冷胶/明胶/纤维素纳米晶体3D支架培养C2C12细胞5天后的细胞骨架染色图，其中A、B分别代表不同比例的结冷胶和明胶。
具体实施方式
以下结合附图和实施例，对本发明进行进一步的详细说明。
1.结冷胶/明胶3D支架的制造：
（1）将结冷胶、明胶溶于水中分别得到浓度为0.5~3%、0.5~5%的结冷胶溶液和明胶溶液；
（2）将结冷胶、明胶溶液以质量比1:5~5:1混合；
（3）混合溶液保持40℃以上温度加入模具，室温放置反应10 min以上得到3D支架。
2.在制造的结冷胶/明胶3D支架上进行骨骼肌细胞培养：
（1）C2C12小鼠成肌细胞复苏离心并重悬于含10％FBS的DMEM/F-12培养基中，于37°C、5% CO2培养箱中培养至细胞汇合达到约80%时，通过胰蛋白酶消化和离心获得细胞悬液；
（2）向细胞悬液中加入培养基混合均匀后接种于3D支架上，在37°C、5% CO2培养箱中培养；
（3）在培养3天后将支架进行活/死细胞染色，并使用荧光显微镜进行观察，绿色为活细胞，红色为死细胞；
（4）培养5天后将支架进行细胞骨架染色，并使用激光共聚焦显微镜进行观察，蓝色为细胞核，红色为细胞骨架。
3.在制造的结冷胶/明胶3D支架上进行小鸡骨骼肌卫星细胞培养：
（1）小鸡骨骼肌卫星细胞复苏离心并重悬于含20％FBS的DMEM/F-12培养基中，于37°C、5% CO2培养箱中培养至细胞汇合达到约80%时，通过胰蛋白酶消化和离心获得细胞悬液；
（2）向细胞悬液中加入培养基混合均匀后接种于3D支架上，在37°C、5% CO2培养箱中培养；
（3）培养5天后将支架进行细胞骨架染色，并使用激光共聚焦显微镜进行观察，蓝色为细胞核，红色为细胞骨架。
4.在制造的结冷胶/明胶/纤维素纳米晶体3D支架上进行骨骼肌细胞3D培养：
（1）将纤维素纳米晶体（CNC）分散于水中得到浓度为0.3~3%的CNC溶液，将明胶溶于CNC溶液中得到浓度为0.5~5%的含有CNC的明胶溶液，将结冷胶溶于水中得到浓度为0.5~3%的结冷胶溶液；
（2）C2C12小鼠成肌细胞复苏离心并重悬于含10％FBS的DMEM/F-12培养基中，于37°C、5% CO2培养箱中培养至细胞汇合达到约80%时，通过胰蛋白酶消化和离心获得细胞悬液；
（3）将结冷胶、含有CNC的明胶溶液以质量比1:5~5:1混合；
（4）将步骤（2）获得的细胞悬液加入步骤（3）获得的混合溶液中混合均匀，室温放置反应10 min以上，加入培养基后于37°C、5% CO2培养箱中培养；
（5）培养5天后将支架进行细胞骨架染色，并使用激光共聚焦显微镜进行观察，蓝色为细胞核，红色为细胞骨架。
Claims (4)
1.一种用于细胞培养肉的可食用的结冷胶/明胶3D支架的制造方法，其特征在于，所述方法包括以下步骤：
（1）将结冷胶和明胶以一定浓度溶解于水中，通过静电相互作用形成水凝胶；
（2）将明胶溶液和结冷胶溶液以一定质量比均匀混合；
（3）在一定温度条件下将混合后得到的溶液以一定质量加入模具中；
（4）将加有混合溶液的模具于室温下放置反应一定时间。
2.根据权利要求1所述的方法，其特征在于，所述结冷胶浓度为0.5~3%，所述明胶浓度为0.5~5%；所述明胶溶液和结冷胶溶液的质量比为1:5~5:1。
3.根据权利要求1所述的方法，其特征在于，所述混合溶液在加入模具时温度保持在40℃以上；所述混合溶液在室温下放置反应10 min以上。
4.根据权利要求1所述的方法，其特征在于，所述结冷胶和明胶均为食品级物料，且已实现商业化生产；所述可食用3D支架的制造方法简单、绿色、高效，无需添加任何有毒交联剂，无需使用冷冻干燥即可直接用于细胞培养，可降低成本并具有良好的细胞粘附性和相容性，适用于细胞培养肉等组织工程领域。
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