High‐Definition Single‐Cell Printing Cell‐by‐Cell Fabrication of Biological Structures

High‐Definition Single‐Cell Printing Cell‐by‐Cell Fabrication of Biological Structures

  一种高精准度的单细胞打印技术。

Introduction

  当前修复受损组织的方法主要是利用物理或药物干预来增强身体的自然愈合特性。然而,当损伤无法修复时,唯一的选择就是移除或替换组织,但由于寻找合适供体可能的免疫排斥需要持续的免疫抑制的挑战,它们可能不是最优选择。组织工程试图创造用于治疗的人工组织和器官,同时也可能带来更好的结果,包括在更长的时间和更少的药物支持下更有效地发挥作用的组织。

  人工组织合成通常使用两种方法:

  • “自下而上”:用多能细胞培养生物结构。
  • “自上而下”:物理将细胞组装为功能单元。

  生物打印技术可以将特定的基块组装成“自下而上”的方法所不能创造的结构。然而,当前的生物打印机在控制细胞组织方面的能力有限,它们不能精确控制细胞-细胞之间的位置。在大的空间尺度上,真实组织的结构和功能是复杂的,单个细胞类型被精确地组织成复杂的结构如组织和器官。因此,无法打印位置和组成可控的密集细胞的结构,是生物打印材料模拟真实组织的形式和功能的主要障碍。为了制造出具有模仿生物系统复杂特性的新一代生物材料,需要一种快速、精确的单细胞打印新方法。

  目前用于装配具有可控细胞尺度特性的生物结构的方法依赖于介导细胞相互作用。例如,使用DNA来控制细胞聚集,或将DNA打印到基质上,使细胞在所需位置聚集。然而,用这些方法建造复杂的多层结构还有待完成。

  其中一种方法是将3D打印与荧光激活细胞分选(FACS)的细胞选择性结合。然而,将细胞选择性集成到生物打印的方法是笨拙和缓慢的,而流式细胞仪由于有大型静电分选器的存在,出口难以接近基板,因此不能以微米级的分辨率打印细胞。压电装置同理。

  本文中提出了一种高精准度的单细胞打印(HD-SCP)方法。通过一种高度微型的微流控分选机完成,可以从候选的混合物中选择所需的单细胞,并以≈10 μm的精度和≈100 Hz的速率打印。其细胞从分选机移动到基质的距离≈3 mm。

Figure 1. Schematic of HD-SCP printing process

  HD‐SCP打印工艺示意图。HD‐SCP依靠微流控喷头将含有细胞的液滴喷射到空气中,随后由荧光激活的分类系统进行检测。具有所需单细胞的液滴通过DEP分类器选择,以打印细胞阵列、图案和明确的细胞球体。

奥内佐格数(Ohnesorge numbers, Oh*)是用于描述液滴喷射行为的无量纲常数,涉及液体黏度、表面张力和密度。其倒数Z = *Oh−1 = (ρσ2R)1/2/μ。

REIS提出:当1 < Z < 10 时,会有稳定的液滴形成。如果Z < 1 时,液体黏度为主要的影响因素,这会降低液滴的初始速度,要保证初始速度不变就需要较大的压力来克服黏性耗散;如果Z > 10 时,会使微滴在喷射前发生膨胀, 产生的液滴会伴随着不必要的卫星滴。Daniele等提出为了确保成功喷出墨滴,必须在1 < Z < 14区间内精确调整墨水成分和打印参数。

Figure 2. HD-SCP printhead design and operation

  • (a)HD‐SCP打印头整合了液滴发生器、荧光液滴分选器和废液收集模块(左)。液滴通过3D空气/液体共流雾化(右)。
  • (b)电极上的恒定电压会产生DEP力,将液滴偏转到废液收集管中。
  • (c)分选系统检测到目标细胞会在短时间内触发电极上的电压中断,以使细胞沉积在基板上。
  • (d)未分选的液滴遵循泊松统计,其中含约10%的细胞。
  • (e)分选后的液滴含细胞率为99.5%。

PMT:PhotoMultiplier Tube,光电倍增管。

FPGA:Field Programmable Gate Array,现场可编程逻辑门阵列,属于专用集成电路中的一种半定制电路,是可编程的逻辑列阵。

AMP:Amplifier,放大器,把输入讯号的电压或功率放大的装置。

Figure 3. HD-SCP enables single cells sorting with high accuracy and high viability

  • (a)500 Hz和1000 Hz的液滴形成频率下,含细胞的液滴的分选精度大于99%。对钙黄绿素和钙黄绿素红混合染色的细胞的针对性分选精度大于98%。错误分类往往是由有限的光学灵敏度引起的。
  • (b)分选后观察到平均细胞活力约为94%,与对照细胞无显着差异。
  • (c)增殖试验表明,分选细胞和对照细胞在4天内的生长速率相似。

Figure 4. HD-SCP selectively printing of single cells

  • (a)每个位置分别有1个/5个/1~5变动的液滴阵列。
  • (b)对打印液滴的测量表明,当打印距离小于6 mm时,平均打印精度约为≈8 µm。
  • (c)从左到右:交替的单细胞,绿色和红色染色的细胞对,交替的5个细胞组以及基于空间变化程序打印的细胞组(从左到右,绿色染色细胞递增;从上到下,红色染色细胞递增)。
  • (d)以不同钙黄绿素染色的细胞密度的生物墨水印刷的“UCSF”图案。
  • (e)钙黄绿素、钙黄绿素红和CellTrace Far Red(呈蓝色表示,易于区分图像)的染色细胞悬浮液中打印的图案。
  • (f)具有绿色细胞和红色细胞交替结构的逐层打印。
  • (g)通过单个细胞打印构建的“埃菲尔铁塔”。

Figure 5. Controlled spheroid formation by HD-SCP

  • (a)通过可控制的细胞数或细胞类型比,可控地形成细胞球的示意图。
  • (b)初始打印细胞数为10(上)和200(下)形成的均匀细胞球。
  • (c)通过打印和被动加载形成细胞球大小的小提琴图。分布越窄代表越均匀,由此HD‐SCP法形成的细胞球更均匀。
  • (d)不同起始细胞数产生的代表性的生物打印细胞球。
  • (e)不同的初始细胞比的生物打印多细胞球。
  • (f)动态控制细胞球形成的示意图。
  • (g)通过打印200个细胞,培养一天后在形成的球状体的一侧打印不同数量的第二种细胞,形成的多细胞两面球体。

Discussion

  • 使用了空气作为周围相而不是油,无需特定基板性质。
  • 单细胞分辨率可控地优化初始细胞条件形成细胞球,并研究其特性。
  • 有原位打印的潜能。

Reference

Zhang P, Abate A R. High-Definition Single-Cell Printing: Cell-by-Cell Fabrication of Biological Structures[J]. Advanced Materials, 2020, n/a(n/a): 2005346.

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