CN114456933A - 一种三维流动培养装置、系统及分析方法 - Google Patents
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Abstract
本发明提供一种三维流动培养装置、系统及分析方法,属于生物医药技术领域。通过结合细胞培养孔板和微流控技术,实现自动化和动态地向孔板内运输营养物质、生化因子和药物溶液,构建孔板内组织和类器官的三维流动培养微环境;通过流体泵系统编程控制实现多个孔板内培养环境的精准调控,有效提高组织和类器官的培养通量和药物检测通量;该装置和系统具有良好的可拓展性和兼容性,能实现显微镜下的培养和实时观测,并利用算法和软件同步分析组织、类器官的生长情况;装置加工采用3D打印和翻模法相结合,加工方法简单且成本低廉。本发明所提出的装置、系统及药物评估方法有望被广泛运用到新药物研发、临床药物筛选以及基础生物医学研究当中。
Description
技术领域
本发明属于生物医药技术领域,是结合细胞培养孔板和微流控技术,构建三维流动微环境,实现组织、类器官和线虫等生物体的高通量和长期培养,以及自动化和动态的药物检测。
背景技术
恶性肿瘤的药物化疗治疗过程复杂,通常需要多种抗癌药物进行组合后在不同时间点顺序使用。为了提高化疗效果,亟需一种能够针对肿瘤病人个体实现精准预测候选药物种类、组合方式、用药剂量和时间的有效性和肿瘤耐药性的方法。
最近研究表明,将患者活检或手术提取得到的肿瘤细胞或组织,在体外三维微环境中培养形成肿瘤组织或类器官,能高度模拟患者原发肿瘤特征和异质性,有望作为新型体外模型用于进行精准药物检测与筛选。
使用肿瘤组织和类器官进行药物测试需要实现高通量和低成本培养、以及精准和动态给药两个主要内容。目前,国内外常用的肿瘤类器官培养方法包括Transwell法、三维基质胶法和悬浮搅动培养法。最近一些生物医药公司(如STEMCELL Technology和Corning等)改进了多孔板结构和材料,推出了适合组织和类器官培养的孔板产品。但这些方法存在的一个主要缺陷是无法模拟体内肿瘤组织所处的动态流动微环境,无法为组织和类器官提供持续的营养物质和氧气交换,以及清除代谢废物,因而无法实现长期的培养和自动化的药物递送。近些年兴起的微流控技术为构建精确可控的、动态的肿瘤类器官培养微环境提供了可能性。但由于微流控装置中流道和空腔尺寸限制了肿瘤类器官的生长,因此无法实现长期培养。同时,微流控装置的加工和实验操作复杂,难以进行规模化的应用。
发明内容
针对现有技术存在的问题,本发明拟结合细胞培养孔板和微流控技术构建高通量的三维流动体外培养装置。该培养装置利用微流道硅胶盖向孔板中输送溶液,利用多通道直流泵控制液体流动,实现自动化和精准递送生化因子和营养物质,并进行动态化的免疫荧光染色和药物检测;通过系统中的显微镜对培养物质进行实时观测和采集图像;最后通过计算机进行图像处理,分析和研究所培养物质的状态和药物的作用效果。
本发明的技术方案:
一种三维流动培养装置,所述的三维流动培养装置3包括流道层3-1、孔层3-2和培养孔板3-3;流道层3-1包括依次连接的液体入口3-1-1、液体分流通道3-1-2、灌注流道模块3-1-3、液体流出通道3-1-5和液体出口3-1-6;灌注流道模块3-1-3上包括n×m个灌注流道单元3-1-4;孔层3-2上包括n+1×m个灌注孔单元3-2-1;培养孔板3-3上包括n+1×m个孔单元。
进一步的,溶液通过液体入口3-1-1进入流道层3-1,首先经过分流通道3-1-2,然后通过第i行第j列1≤i≤n,1≤j≤m个灌注流道单元3-1-4进入孔层3-2,经灌注孔单元3-2-1上的大孔进入培养孔板3-3的一个孔单元中,最后由灌注单元3-2-1上的小孔流出孔层3-2,回到流道层3-1中第i行第j+1列的灌注流道单元3-1-4,最后经过液体流出通道3-1-5,从液体出口3-1-6流出。灌注单元3-2-1上的小孔半径为r2,灌注单元3-2-1上的大孔半径为r1。
进一步的,所述的灌注流道单元3-1-4中流道宽度逐渐由小变大,且两端形状为半圆圆弧。所述的灌注孔单元3-2-1为一半径为r的柱形凸起,柱形凸起中贯通设置两个孔,其中一个孔的半径与灌注流道单元3-1-4中流道宽度大的端部半圆圆弧的半径r1相同,另一个孔的半径与灌注流道单元3-1-4中流道宽度小的端部半圆圆弧的半径r2相同。柱形凸起插入所述孔板3-3的孔单元中,且柱形凸起端面与孔单元底面留有空间,为待培养的物质提供培养空间。
进一步的,流道层3-1的灌注流道单元3-1-4、孔层3-2的灌注孔单元3-2-1和培养孔板3-3的孔单元对准;其中,第i行第j列的灌注流道单元3-1-4半径为r1的半圆圆弧和第i+1行第j列灌注孔单元3-2-1半径为r1的孔的下半圆弧对准,第i行第j列的灌注流道单元3-1-4半径为r2的半圆圆弧和第i行第j列灌注孔单元3-2-1半径为r2的孔的上半圆弧对准,灌注孔单元3-2-1半径为r的柱形凸起和培养孔板3-3的孔单元对准。
进一步的,孔层3-2的灌注孔单元3-2-1的设计方式为如下两种方式之一;
第一种:半径为r1的液体流入孔和半径为r2的液体流出孔的孔深相同,都等于半径为r的柱形凸起高度h2和孔层底面厚度h3之和,即h2+h3;
第二种:半径为r1的液体流入孔的孔深为h2+h3+h4,半径为r2的液体流出孔的孔深仅为h2+h3。h4为流入孔高出柱形凸起的高度;
进一步的,灌注孔单元3-2-1中,参数l1表示培养孔板3-3两个相邻孔中心的距离,柱形凸起的半径r比培养孔板3-3的孔半径大0.5至1毫米,液体流出孔半径r2略小于待培养物质的等效半径,h2+h4小于培养孔板3-3孔的深度;同时,r1、r2、h2、h3和h4的选取需满足能使液体充分充满培养孔板3-3的孔内空间,且不产生紊乱的流动影响待培养物质的生长;r1、r2、h2、h3和h4的数值可通过流体力学仿真和实验测量进行选取和优化;h1的选取由通入液体的通量决定,通量小时h1取值小例如,几十到几百微米,通量大时h1取值可达几百微米至几十毫米。
进一步的,流道层3-1可设计多个液体入口3-1-1,其连接的分流通道3-1-2可根据实际需求设计复杂流道形状如“圣诞树”型,将液体入口与不同的灌注流道单元3-1-4相连,实现向培养孔板3-3的不同孔单元中灌注不同种类或浓度的溶液。利用流体力学中集中参数模型仿真辅助设计灌注流道形状。
进一步的,培养孔板3-3为平底的细胞培养的384孔板,或者PCR 384孔板、96孔板、48孔板;流道层和孔层的设计尺寸包括l1、l2、r1、r2、h1等依据孔板的尺寸进行选择和调整。
一种三维流动培养装置的制作方法,所述的制作方法包括以下四个主要步骤:
步骤一:利用3D打印或其它精密微纳加工技术制作流道层3-1和孔层3-2的阳模,其中流道层3-1和孔层3-2中的流道和通孔在阳模中为凸起区域,而孔层3-2的柱形凸起在阳模中为凹槽;
步骤二:基于阳模利用翻模法制作PDMS流道层3-1和孔层3-2;
步骤三:利用等离子体处理PDMS流道层3-1和孔层3-2表面,在显微镜下进行两层结构对准,键合,并在烘箱内烘烤直至两层PDMS紧密粘合在一起。
步骤四:将键合后构成的PDMS装置与培养孔板3-3组装,使孔层4-2的所有柱形凸起卡入培养孔板3-3的孔中。
一种三维流动培养系统,所述的三维流动培养系统包括细胞培养箱、多通道流体控制泵1、液体灌注系统2、三维流动培养装置3、显微镜和图像采集系统4以及计算机5。
进一步的,由流体控制泵1通过液体灌注系统2可连接很多个三维流动培养装置3,从而实现在很多个孔板中同时进行组织和类器官的培养,有效提高培养通量和药物检测通量。
一种三维流动培养分析方法,具体步骤如下:
步骤一:利用溶液如聚(甲基丙烯酸-2-羟乙酯)poly-HEMA溶液处理培养孔板3-3底面,使形成疏水的、细胞不贴附的表面;
步骤二:向液体灌注系统2中的试管内加入培养物质的溶液或待测药物;
步骤三:向培养孔板3-3中加入待培养的物质,并将流道层3-1和孔层3-2与孔板3-3组装构成三维流动培养装置3;通过导管将液体控制泵1出口和含有溶液的试管口相连,将试管的另一个出口与三维流动培养装置3的流体入口相连,最后将三维流动培养装置3的流体出口和储存废液的试管口相连;
步骤四:将三维流动培养装置3放置于细胞培养箱中进行培养;
步骤五:控制多通道流体控制泵1的运行,进行定时、定量地向三维流动培养装置3中输送培养物质的溶液或待测药物;
步骤六:通过显微镜和图像采集系统4进行待培养的物质的实时观测和图像采集;
步骤七:利用计算机5中的图像分析算法和软件分析培养过程。
所述的待培养的物质包括:悬浮细胞、三维生物组织、类器官、线虫、斑马鱼等生物医学中常用的细胞和动物模型。
本发明的有益效果:1)本发明所设计的三维流动培养装置由细胞培养孔板和双层PDMS硅胶盖构成;细胞培养孔板(如384孔板)是生物医学领域最常用的细胞和组织培养装置,成本低廉;硅胶盖采用翻模法制作,材料为聚二甲基硅氧烷(PDMS),制作流程简单,成本低廉;且硅胶盖与孔板通过卡入式组装,可拆卸,使用方便灵活;2)通过硅胶盖、灌流系统和流体泵能实现编程控制孔板内物质运输和更换,同时避免孔板内悬浮培养的细胞和组织等物质随液体流出孔板,提高了培养流程的自动化和可控性;3)便于实现同时按需向多个孔板内进行营养物质和药物输运,极大地提高了培养通量和药物检测通量;能够实现在不同时间点的有序药物输运和动态药物检测。
附图说明
图1是生物体(如组织和类器官)的三维流动培养系统示意图;
图2是三维流动培养装置的爆炸图,以及装置中重复单元结构和其中液体流动方向的三维示意图;
图3是三维流动培养装置中流道层结构示意图和设计参数;
图4是三维流动培养装置中孔层结构示意图和设计参数;
图5是三维流动培养装置的孔层中灌流孔单元的截面图;其中,(a)为方式一,(b)为方式二;
图6是流动层和孔层的对准示意图;其中,实线表示流道层,虚线表示孔层;
图7(a)是实施例一中所用的赛默飞世尔公司的384孔板及尺寸示意图;
图7(b)和图7(c)是基于COMSOL流体力学仿真得到的一个培养孔内的液体流线;
图1中:1流体控制泵;2灌流系统;3三维流动培养装置;4显微镜和图像采集系统;5计算机。
图2中:3-1流道层;3-2孔层;3-3培养孔板;
图3中:3-1-1液体入口;3-1-2分流通道;3-1-3灌注流道模块;3-1-4灌注流道单元;3-1-5流出通道;3-1-6液体出口;
l1灌注流道单元尺寸;l2流入孔和流出孔间距;r1流入孔半径;r2流出孔半径;h1流道高度。
图4中:3-2-1灌注孔单元;
l1灌注孔单元尺寸;r1流入孔半径;r2流出孔半径;r柱形凸起半径;d1流入孔圆心距离灌注孔单元中心的距离;d2流出孔圆心距离灌注孔单元中心的距离;h2柱形凸起高度;h3孔层底面厚度。
图5中:r1流入孔内半径;r2流出孔内半径;r3流入孔外半径;r柱形凸起半径;h2柱形凸起高度;h3孔层底面厚度;h4流入孔高出柱形凸起的高度。
图6:l1流道层灌注流道单元尺寸和孔层灌注孔单元尺寸。
具体实施方式
下面的实施例将对本发明予以进一步的说明,但并不因此而限制本发明。
实施例1
选取美国赛默飞世尔科技有限公司(Thermo Fisher)的384细胞培养孔板(货号:165195)作为装置第三层。该384孔板的每个孔形状为长方体,尺寸参数如图7(a)所示。设计如图2、图3、图4、图5(中的(a))所示的生物组织和类器官的培养装置;其中受孔板形状影响,PDMS孔层设计长方体形凸起而非柱形凸起,即将图4和5中半径为r的柱形凸起改为长方体形凸起,长方体凸起的横截面为边长r的正方形,凸起的高度为h2;具体设计参数如下表所示。
r<sub>1</sub> | 0.2mm | r<sub>2</sub> | 0.7mm | r | 3.8mm |
d<sub>1</sub> | 2mm | d<sub>2</sub> | 1mm | l<sub>1</sub> | 8.3mm |
h<sub>1</sub> | 0.5mm | h<sub>2</sub> | 7.1mm | h<sub>3</sub> | 3mm |
表1:实施例1中装置的设计参数
液体通过长方体凸起上半径为r2的孔流入孔板中,然后从半径为r1的孔流出,重新回到PDMS流道层。基于COMSOL仿真计算可得到孔板中液体的流动情况,如图7(b)所示;液体能够充满孔板(包括到达孔的底部),且流动较为平稳,因此上述设计参数基本符合要求。
实施例2
仍选取美国赛默飞世尔科技有限公司(Thermo Fisher)的384细胞培养孔板(货号:165195)作为装置第三层。设计如图2、图3、图4、图5(中的(b))所示的生物组织和类器官的培养装置;相较于实施例1,将液体流入孔的外围孔壁延伸至靠近孔板底部的位置,从而使液体能够直达孔底,更好的充满整个孔板。此时,设计参数h2=3mm和h4=9mm,其它参数与实施例1相同。
基于COMSOL仿真计算可得到孔板中液体的流动情况,如图7(c)所示,此时流动更加平稳且液体能充分充满孔板,能起到较好的孔板内外物质运输效果。
Claims (10)
1.一种三维流动培养装置,其特征在于,所述的三维流动培养装置(3)包括流道层(3-1)、孔层(3-2)和培养孔板(3-3);流道层(3-1)包括依次连接的液体入口(3-1-1)、液体分流通道(3-1-2)、灌注流道模块(3-1-3)、液体流出通道(3-1-5)和液体出口(3-1-6);灌注流道模块(3-1-3)上包括n×m个灌注流道单元(3-1-4);孔层(3-2)上包括(n+1)×m个灌注孔单元(3-2-1);培养孔板(3-3)上包括(n+1)×m个孔单元;
所述的灌注流道单元(3-1-4)中流道宽度逐渐由小变大,且两端形状为半圆圆弧;所述的灌注孔单元(3-2-1)为一半径为r的柱形凸起,柱形凸起中贯通设置两个孔,其中一个孔的半径与灌注流道单元(3-1-4)中流道宽度大的端部半圆圆弧的半径r1相同,另一个孔的半径与灌注流道单元(3-1-4)中流道宽度小的端部半圆圆弧的半径r2相同;柱形凸起插入所述孔板(3-3)的孔单元中,且柱形凸起端面与孔单元底面留有空间,为待培养的物质提供培养空间;
所述的流道层(3-1)的灌注流道单元(3-1-4)、孔层(3-2)的灌注孔单元(3-2-1)和培养孔板(3-3)的孔单元对准;其中,第i行第j列的灌注流道单元(3-1-4)半径为r1的半圆圆弧和第(i+1)行第j列灌注孔单元(3-2-1)半径为r1的孔的下半圆弧对准,第i行第j列的灌注流道单元(3-1-4)半径为r2的半圆圆弧和第i行第j列灌注孔单元(3-2-1)半径为r2的孔的上半圆弧对准,灌注孔单元(3-2-1)半径为r的柱形凸起和培养孔板(3-3)的孔单元对准。
2.根据权利要求1所述的一种三维流动培养装置,其特征在于,溶液通过液体入口(3-1-1)进入流道层(3-1),首先经过分流通道(3-1-2),然后通过第i行第j列个灌注流道单元(3-1-4)进入孔层(3-2),经灌注孔单元(3-2-1)上的大孔进入培养孔板(3-3)的一个孔单元中,最后由灌注单元(3-2-1)上的小孔流出孔层(3-2),回到流道层(3-1)中第i行第(j+1)列的灌注流道单元(3-1-4),最后经过液体流出通道(3-1-5),从液体出口(3-1-6)流出;灌注单元(3-2-1)上的小孔半径为r2,灌注单元(3-2-1)上的大孔半径为r1。
3.根据权利要求1所述的一种三维流动培养装置,其特征在于,孔层(3-2)的灌注孔单元(3-2-1)的设计方式为如下两种方式之一;
第一种:半径为r1的液体流入孔和半径为r2的液体流出孔的孔深相同,都等于半径为r的柱形凸起高度h2和孔层底面厚度h3之和,即h2+h3;
第二种:半径为r1的液体流入孔的孔深为h2+h3+h4,半径为r2的液体流出孔的孔深仅为h2+h3;h4为流入孔高出柱形凸起的高度。
4.根据权利要求1所述的一种三维流动培养装置,其特征在于,灌注孔单元(3-2-1)中,参数l1表示培养孔板(3-3)两个相邻孔中心的距离,柱形凸起的半径r比培养孔板(3-3)的孔半径大0.5至1毫米,液体流出孔半径r2略小于待培养物质的等效半径,h2+h4小于培养孔板(3-3)孔的深度;同时,r1、r2、h2、h3和h4的选取需满足能使液体充分充满培养孔板(3-3)的孔内空间,且不产生紊乱的流动影响待培养物质的生长;r1、r2、h2、h3和h4的数值可通过流体力学仿真和实验测量进行选取和优化;h1的选取由通入液体的通量决定。
5.根据权利要求1所述的一种三维流动培养装置,其特征在于,流道层(3-1)可设计多个液体入口(3-1-1),其连接的分流通道(3-1-2)可根据实际需求设计复杂流道形状,将液体入口与不同的灌注流道单元(3-1-4)相连,实现向培养孔板(3-3)的不同孔单元中灌注不同种类或浓度的溶液;利用流体力学中集中参数模型仿真辅助设计灌注流道形状。
6.根据权利要求1所述的一种三维流动培养装置,其特征在于,培养孔板(3-3)为平底的细胞培养的384孔板,或者PCR 384孔板、96孔板或48孔板;流道层和孔层的设计尺寸依据孔板的尺寸进行选择和调整。
7.一种三维流动培养系统,其特征在于,所述的三维流动培养系统包括细胞培养箱、多通道流体控制泵(1)、液体灌注系统(2)、权利要求1-6任一所述的三维流动培养装置(3)、显微镜和图像采集系统(4)以及计算机(5)。
8.根据权利要求7所述的一种三维流动培养系统,其特征在于,由流体控制泵(1)通过液体灌注系统(2)可连接很多个三维流动培养装置(3),从而实现在很多个孔板中同时进行组织和类器官的培养,有效提高培养通量和药物检测通量。
9.一种采用权利要求7-8任一所述的三维流动培养系统进三维流动培养分析方法,其特征在于,具体步骤如下:
步骤一:利用溶液处理培养孔板(3-3)底面,使形成疏水的、细胞不贴附的表面;
步骤二:向液体灌注系统(2)中的试管内加入培养物质的溶液或待测药物;
步骤三:向培养孔板(3-3)中加入待培养的物质,并将流道层(3-1)和孔层(3-2)与孔板(3-3)组装构成三维流动培养装置(3);通过导管将液体控制泵(1)出口和含有溶液的试管口相连,将试管的另一个出口与三维流动培养装置(3)的流体入口相连,最后将三维流动培养装置(3)的流体出口和储存废液的试管口相连;
步骤四:将三维流动培养装置(3)放置于细胞培养箱中进行培养;
步骤五:控制多通道流体控制泵(1)的运行,进行定时、定量地向三维流动培养装置(3)中输送培养物质的溶液或待测药物;
步骤六:通过显微镜和图像采集系统(4)进行待培养的物质的实时观测和图像采集;
步骤七:利用计算机(5)中的图像分析算法和软件分析培养过程。
10.根据权利要求9所述的分析方法,其特征在于,所述的待培养的物质包括生物医学中的细胞和动物模型。
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