seeding cd34+ cells into fluidic chips

التمايز والتحفيز الميكانيكي للخلايا البطانية المشتقة من hiPSC

During embryonic development, the heart is the first organ to establish functionality1, with detectable contractions from the earliest stage of endocardial tube formation2. Circulation, along with the mechanical cues mediated by the flow of blood within the vessel, controls many crucial aspects of early development. Prior to fetal circulation establishment, the vasculature is organized into a primary capillary plexus; upon cardiac functioning, this plexus reorganizes into venous and arterial vasculature3. The role of mechanical cues in arteriovenous specification is reflected by the pan-endothelial expression of arterial and venous markers before blood flow initiation4.
Hemodynamic forces not only control the development of the vasculature itself but also play a fundamental role in the control of blood cell formation. Hematopoietic stem and progenitor cells (HSPCs) emerge from specialized endothelial cells called hemogenic endothelium5,6,7,8, present in different anatomical regions of the embryos exclusively in the early stage of development. Heart-deficient models, together with in vitro models, have demonstrated that mechanical cues instruct and increase HSPC production from the hemogenic endothelium9,10,11,12,13,14.
Different types of flow dynamics have been shown to differentially control the cell cycle15, known to be important in both hemogenic endothelium16,17 and arterial cell specification18. Altogether, mechanical cues are critical determinants of cell identity and function during development. Novel in vitro fluidic devices allow us to overcome the limitations involved with studying developmental mechanobiology during human blood development in vivo.
The overall goal of the protocol in this manuscript is to describe, step-by-step, the experimental pipeline to study the effect of shear stress on human endothelial cells derived in vitro from human induced pluripotent stem cells (hiPSCs). This protocol contains detailed instructions on the differentiation of hiPSCs into endothelial cells and their subsequent seeding into fluidic chips for the stimulation protocol. Using this, different in vitro-derived endothelial cells can be tested for their ability to sense the shear stress by analyzing their orientation in response to the flow. This will allow other laboratories to address questions about the response to shear stress and its functional consequences on different endothelial cell identities.

تسليط الضوء على المؤلف: دراسة الخلايا البطانية المشتقة من hiPSC المزروعة تحت التحفيز الميكانيكي بوساطة السوائل 

في المختبر نموذج لسفينة بشرية جنينية على الرقاقة لدراسة علم الأحياء الميكانيكي التنموي

Our research explores how mechanical cues instruct the fate of endothelial cells during development, for which we combine human-induced pluripotent stem cell differentiation with fluidic-mediated mechanical stimulation. Endothelial cells are constantly exposed to the movement of blood, so important signals are missed out in static culture, which makes this the main challenge in studying endothelial cell biology using traditional culture systems. Mechanical cues, in fact, activate many crucial signaling pathways that might be overlooked in cells cultured in static conditions.Our protocol provides an easy, step-by-step guide on how to differentiate and mechanically stimulate induced pluripotent stem-cell-derived endothelial cells. This will help many laboratories in adopting a thermic culture to easily address questions about endothelial cell behavior while under flow.

Watch this Scientific Journal Video about Seeding CD34+ Cells into Fluidic Chips at JoVE.com

Seeding CD34+ Cells into Fluidic Chips  

بذر خلايا CD34 + في رقائق السوائل

للبدء ، قم بالطرد المركزي للخلايا الإيجابية CD34 المعزولة وإعادة تعليق حبيبات الخلية في 300 ميكرولتر من وسط SFM-34 الذي تم تسخينه مسبقا والذي يتم استكماله ب 4 سيتوكينات مختلطة ، وعد الخلايا باستخدام 10 ميكرولتر من تعليق الخلية. بعد عد الخلايا وإعادة تعليقها في حجم نهائي يبلغ 150 ميكرولتر من SFM-34 المكمل بالسيتوكين ، أضف 0.5 ميكرولتر من مثبط ROCK إليها. قم بشفط الصفيحة ببطء من رقائق السوائل عن طريق وضع طرف ماصة P200 داخل الخزان على حافة القناة.ثم أضف تعليق الخلية إلى القناة باستمرار ، مما يضمن عدم تكوين فقاعات. احتضان الشريحة طوال الليل عند 37 درجة مئوية و 5٪ ثاني أكسيد الكربون لربط الخلايا بالقناة تماما. عندما تكون الخلايا البطانية متصلة بالكامل ، قم بنضح الوسط كما هو موضح سابقا.أضف 200 ميكرولتر من SFM-34 المكمل بالسيتوكين إلى الشريحة. استبدل الوسط يوميا حتى تصل الخلايا إلى التقاء 90 إلى 100٪.

Research

JoVE Journal

Developmental Biology

Seeding CD34+ Cells into Fluidic Chips

262 Views.  University of Edinburgh. للبدء ، قم بالطرد المركزي للخلايا الإيجابية CD34 المعزولة وإعادة تعليق حبيبات الخلية في 300 ميكرولتر من وسط SFM-34 الذي تم تسخينه مسبقا والذي يتم استكماله ب 4 سيتوكينات مختلطة ، وعد الخلايا باستخدام 10 ميكرولتر من تعليق الخلية. بعد عد الخلايا وإعادة تعليقها في حجم نهائي يبلغ 150 ميك...

Video: Seeding CD34+ Cells into Fluidic Chips  

In Vitro Model of Fetal Human Vessel On-chip to Study Developmental Mechanobiology

The heart is the first organ to be functionally established during development, thus initiating blood circulation very early in gestation. Besides transporting oxygen and nutrients to ensure fetal growth, fetal circulation controls many crucial developmental events taking place within the endothelial layer through mechanical cues. Biomechanical signals induce blood vessel structural changes, establish arteriovenous specification, and control the development of hematopoietic stem cells. The inaccessibility of the developing tissues limits the understanding of the role of circulation in early human development; therefore, in vitro models are pivotal tools for the study of vessel mechanobiology. This paper describes a protocol to differentiate endothelial cells from human induced pluripotent stem cells and their subsequent seeding into a fluidic device to study their response to mechanical cues. This approach allows for long-term culture of endothelial cells under mechanical stimulation followed by&#160;retrieval of the endothelial cells for phenotypical and functional characterization. The in vitro model established here will be instrumental to elucidate&#160;the intracellular molecular mechanisms that transduce the signaling mediated by mechanical cues, which ultimately orchestrate vessel development during human fetal life.

Described here is a simple workflow to differentiate endothelial cells from human pluripotent stem cells followed by a detailed protocol for their mechanical stimulation. This allows for the study of the developmental mechanobiology of endothelial cells. This approach is compatible with downstream assays of live cells collected from the culture chip after mechanical stimulation.

The heart is the first organ to be functionally established during development, thus initiating blood circulation very early in gestation. Besides ...

﻿Differentiation of hiPSCs into Endothelial Cells

Differentiation of hiPSCs into Endothelial Cells  

To begin, on day zero, pre-warm serum-free differentiation medium or SFD medium containing Mix 1 cytokine to 37 degrees Celsius. Then, under a sterile tissue culture hood, add the Mix 1 cytokine-supplemented medium to each well of a cell repellent six-well plate. To prepare the cells, aspirate the culture medium from a six-well plate containing human-induced pluripotent STEM cells or human IPS cells.Wash them with DPBS followed by aspirating the DPBS. Then, add the dissociation reagent to the cells and incubate them at room temperature for a minute. After aspirating this dissociation reagent, incubate the cells for another three minutes at room temperature.Then, tap the plate containing the cells 10 times on each side to detach the cell clusters. Add one milliliter of pre-warmed Mix 1 cytokine-supplemented SFD medium to the cells per well. Using a pasture pipette, transfer the cell clusters from each well to one well of the cell repellent plate containing the medium with Mix 1 cytokine.After introducing the plate into the incubator, move it back and forth, as well as side to side, to disperse the content evenly and incubate it for embryoid bodies, or EB, formation. The next day, swirl the cell repellent plate containing the EBs to collect them into the center of the wells. Using a pasteur pipette, transfer the EB suspension from the wells to a 15-milliliter centrifuge tube.Wait five to 10 minutes for the EBs to settle at the bottom of the tube. Meanwhile, wash the cell repellent plates with sterile water or DPBS to remove single cells or debris. Add two milliliters of Mix 1 cytokine-supplemented SFD medium to each well of the plate.Now, gently aspirate the supernatant from the centrifuge tube without dislodging the EBs. Resuspend the EBs in the calculated volume of SFD medium containing Mix 1 cytokine. Add one milliliter of EB suspension to each well of the washed cell repellent plate already containing two milliliters of medium.Incubate the plate after dispersing the EBs by gently moving the plate as mentioned before. After gathering the EBs at the center of the wells, add the chiron on the side of each well, avoiding direct contact with the cells, Incubate the wells as demonstrated previously. On days three and six of differentiation, collect the EBs and perform media changes as demonstrated previously for day one.Use SFD medium supplemented with Mix 3 cytokines on day three and use SFD medium supplemented with Mix 4 cytokines on day six. After collecting and settling the EBs from the cell repellent plate into a 15-milliliter centrifuge tube, as demonstrated previously, aspirate the supernatant, then add one milliliter of cell dissociation reagent per well of EB collected in the tube to resuspend the EBs. Next, transfer one milliliter of this EB suspension to each well of the cell repellent plate washed with water.After incubating the plate for 10 minutes in the incubator, use a P1000 pipette to dissociate the EBs in each well by gently pipetting up and down no more than 10 times. Then, add five milliliters of washing buffer per well of dissociated EBs. Collect the cells into a 50-milliliter centrifuge tube by passing them through a 40-micron strainer.After counting the cells in the filtered suspension, spin the cells down for 10 minutes at 300g. Resuspend the cells in 300 microliters of washing buffer by gently pipetting a few times, ensuring no clumps are present. Proceed to isolate the CD34-positive cells using a CD34 microbead kit per the manufacturer's instructions.EBs of good quality showed a defined edge by day two of the differentiation and appeared clear and bright when observed under a microscope. The presence of darker areas indicated cell death within the EBs. Following chiron treatment with varying concentrations on day two, quantifying the number of CD34-positive cells at day eight of differentiation revealed the response of the cell line to the treatment.Flow cytometry showed that the CD34-positive enrichment using the magnetic beads provided about 85%CD34-positive cells.

تمايز hiPSCs إلى خلايا بطانية

To begin, centrifuge the isolated CD34-positive cells and resuspend the cell pellet in 300 microliters of pre-warmed SFM-34 medium that is supplemented with mixed 4 cytokines, and count the cells using 10 microliters of the cell suspension. After counting and resuspending the cells in a final volume of 150 microliters cytokine-supplemented SFM-34, add 0.5 microliters of ROCK inhibitor to it. Slowly aspirate the laminin from fluidic chips by placing the tip of a P200 pipette inside the reservoir on the edge of the channel.Then add the cell suspension into the channel consistently, ensuring no bubbles are formed. Incubate the chip overnight at 37 degrees Celsius and 5%carbon dioxide to attach the cells to the channel completely. When the endothelial cells are fully attached, aspirate the medium as described previously.Add 200 microliters of cytokine-supplemented SFM-34 into the chip. Replace the medium daily until the cells reach 90 to 100%confluency.

Application of Continuous Flow to Endothelial Cells: Aorta-on-a-Chip

Install the perfusion set into the unit per the manufacturer's protocol. Inside a hood, attach an empty fluidic chip to it and add cytokine supplemented SFM-34 medium to fill both reservoirs under sterile conditions. Transfer the fluidic unit to the incubator, connecting it to the pump for bubble removal and calibration.Carefully remove the fluidic unit with the connected set from the incubator and transfer it into the hood along with the chips containing the cells. Clamp the tubing on both sides of the test chip. Remove the clamped tubing from the test chip and connect the chip containing the cells to the tubing.After removing or opening the clamps, transfer the system to the incubator and connect the air pump to the fluidic unit. Remove the fluidic unit from the pump and move it into the hood. Clamp the tubing on both sides of the chip and remove the tubing from the reservoirs on the chip.After gently removing the medium from the chip, Wash the cells with Dulbecco's Phosphate-Buffered Saline or DPBS. Add 150 microliters of dissociation buffer and incubate for three minutes at 37 degrees Celsius. When the cells are detached, collect the dissociation buffer from one reservoir and wash the channel once with DPBS.Wash the reservoir with the washing buffer to collect the remaining cells from the chip. Finally, add one milliliter of washing buffer to the collected cells before using 10 microliters from it to count the cells. Before the stimulation, cells showed random orientation, but with stimulation, they reoriented parallel to the direction of the flow.CD34 expression profile of cells cultured under flow for five days and percentage of CD34 positive cells retrieved from the fluidic channel, showed the effectiveness of the protocol.