Human Liver Spheroids from Peripheral Blood for Liver Disease Studies

요약

Here we present a non-genetic method to generate human autologous liver spheroids using mononuclear cells isolated from steady-state peripheral blood.

초록

Human liver cells can form a three-dimensional (3D) structure capable of growing in culture for some weeks, preserving their functional capacity. Due to their nature to cluster in the culture dishes with low or no adhesive characteristics, they form aggregates of multiple liver cells that are called human liver spheroids. The forming of 3D liver spheroids relies on the natural tendency of hepatic cells to aggregate in the absence of an adhesive substrate. These 3D structures possess better physiological responses than cells, which are closer to an in vivo environment. Using 3D hepatocyte cultures has numerous advantages when compared with classical two-dimensional (2D) cultures, including a more biologically relevant microenvironment, architectural morphology that reassembles natural organs as well as a better prediction regarding disease state and in vivo-like responses to drugs. Various sources can be used to generate spheroids, like primary liver tissue or immortalized cell lines. The 3D liver tissue can also be engineered by using human embryonic stem cells (hESCs) or induced pluripotent stem cells (hiPSCs) to derive hepatocytes. We have obtained human liver spheroids using blood-derived pluripotent stem cells (BD-PSCs) generated from unmanipulated peripheral blood by activation of human membrane-bound GPI-linked protein and differentiated to human hepatocytes. The BD-PSCs-derived human liver cells and human liver spheroids were analyzed by light microscopy and immunophenotyping using human hepatocyte markers.

서문

In recent years three-dimensional (3D) spheroid culture systems have become an important tool to study various areas of cancer research, drug discovery, and toxicology. Such cultures raise great interest because they bridge the gap between two-dimensional (2D) cell culture monolayers and complex organs¹.

In the absence of an adhesive surface, compared to the 2D cell culture, the formation of spheroids is based on the natural affinity of these cells to cluster in 3D form. These cells organize themselves into groups consisting of one or more types of mature cells. Free of foreign materials, these cells interact with each other like in their original microenvironment. The cells in 3D culture are much closer and have a proper orientation toward each other, with higher extracellular matrix production than 2D cultures, and constitute a close to natural environment ².

Animal models have been used for a long time to study human biology and diseases³. In this regard, there are intrinsic differences between humans and animals, which makes these models not entirely suitable for extrapolative studies. 3D culture spheroids and organoids represent a promising tool to study tissue-like architecture, interaction, and crosstalk between different cell types that occur in vivo and can contribute to reducing or even replacing animal models. They are of particular interest for studying the pathogenesis of liver diseases as well as drug screening platforms⁴.

3D spheroid culture is of particular importance for cancer research as it can eliminate the discontinuity between the cells and their environment by reducing the need for trypsinization or collagenase treatment needed for preparing the tumor cell monolayers for 2D cultures. Tumor spheroids enable the study of how the normal versus malignant cells receive and respond to signals from their surroundings⁵ and are an important part of tumor biology studies.

Compared to the monolayer, 3D cultures consisting of various cell types resemble tumor tissues in their structural and functional properties and therefore are suitable for studying metastasis and invasion of tumor cells. That is why such spheroid models are contributing to accelerating cancer research⁶.

Spheroids are also helping to develop the technology to create human organoids because tissue and organ biology are very challenging to study, particularly in humans. Progress in stem cell culture makes it possible to develop 3D cultures like organoids consisting of stem cells and tissue progenitors as well as different types of mature (tissue) cells from an organ with some functional characteristics like a real organ that can be used to model organ development, diseases, but they also can be considered useful in regenerative medicine⁷.

Primary human hepatocytes are usually used for studying in vitro biology of human hepatocytes, liver function, and drug-induced toxicity. Cultures of human hepatocytes have two main drawbacks, firstly, the limited availability of primary tissue like human hepatocytes, and secondly, the tendency of hepatocytes to rapidly dedifferentiate in 2D culture thereby losing their specific hepatocyte function⁸. 3D hepatic cultures are superior in this regard and have recently been made from differentiated human embryonic stem cells (hESCs) or induced pluripotent stem cells (hiPSCs)⁹. Bioengineered hepatic 3D spheroids are of particular interest to study development, toxicity, genetic and infectious diseases of the liver, as well as in drug discovery for the treatment of liver diseases¹⁰. Lastly, they also have the potential to be used clinically, knowing that acute liver diseases have a mortality rate of nearly 80%, bio-artificial liver and/or hepatic spheroids could potentially rescue these patients by providing partial liver function until a suitable donor can be found¹¹.

We have established a protocol for the generation of human hepatic spheroids using blood-derived pluripotent stem cells (BD-PSCs) to prepare differently sized spheroids containing 4000 to 1 x 10⁶ cells and analyzed them by means of light microscopy and immunofluorescence. We also tested the capability of hepatocyte-specific function, assessing the expression of cytochrome P450 3A4 (CYP3A4) and 2E1 (CYP2E1) enzymes that belong to the cytochrome P450 family that have important roles in cellular and drug metabolism through the process of detoxification¹².

프로토콜

Ethical approval was obtained (ACA CELL Biotech GmbH/25b-5482.2-64-1) for performing these experiments and informed consent was signed by all donors before blood extraction in compliance with institutional guidelines.

1. Preparation of mononuclear cells (MNCs) from human peripheral blood (PB)

1. Extract 30 mL of blood from healthy donors with the help of trained medical personnel according to the standard protocol.
2. Isolate PBMNCs using density gradient media according to the protocol published by Becker-Kojić et al.¹³. Isolate, by pipetting, the interphase layer between plasma and density gradient media and use sterile phosphate buffer saline (PBS) to wash the isolated cells.
3. Use a counting chamber and count the number of cells using standard methods.

2. Dedifferentiation of MNCs upon activation with human GPI-anchored glycoprotein

1. Place 6 x 10⁶ mononuclear cells in PBS/1% bovine serum albumin (BSA) in a polystyrene tube (15 mL) and incubate with the specific antibody for 30 min at 37 °C according to Becker-Kojić et al.¹³.
2. Centrifuge the cells at 300 x g at room temperature and replace PBS/BSA with Iscove's modified Dulbecco's medium supplemented with 10% fetal bovine serum (FBS).
3. Grow the cells in 15 mL polystyrene tubes in a 5% CO₂ incubator at 37 °C for 8-10 days as described in Becker-Kojić et al.¹³. On day 5 (D5), add 1-2 mL of Iscove's medium supplemented with 10% FBS to each 15 mL tube.

3. Sorting of newly generated dedifferentiated cells

1. Centrifuge cultured cell suspension at room temperature for 10 min at 300 x g and aspirate with a sterile pipette the resulting supernatant according to Becker-Kojić et al.¹³.
2. Resuspend pellet obtained by centrifugation in 90 µL of cold PBS buffer (PBS pH 7.2, 0.5 % BSA, and 2 mM EDTA) and add 10 µL of CD45 positive nano-sized magnetic beads and incubate on ice for 15 min.
3. Wash the cell suspension with 2 mL of PBS buffer, centrifuge it at 300 x g for 10 min at room temperature and add 500 µL of PBS buffer to the cells and resuspend thoroughly.
4. Pipette 500 µL of pre-cooled PBS buffer into the column to wash and place it in the magnetic field using a magnetic stand.
5. Wash the cells placed on the column, twice with 500 µL of PBS buffer and collect flow-through containing CD45 negative cells in Iscove's medium supplemented with 10% FBS.
6. Use a counting chamber to determine the number of reprogrammed cells.

4. Preparation of glass coverslips for the generation of human hepatocytes

1. Separate glass coverslips (14 mm) and incubate them in nonionic detergent for 10 min. Wash glass coverslips in deionized water till no bubbles remain and incubate them in 1M HCl for 30 min (adapted from Marchenko et al.¹⁴).
2. Wash glass coverslips with deionized water at least 3x and dry them overnight at room temperature. Autoclave dried glass coverslips in a suitable container.

5. Coating cell culture plates with biolaminin for 2D hepatic differentiation of BD-PSCs

1. Place autoclaved glass coverslips with a sterile pair of tweezers in 4 well plates and turn UV lights on for 30 min to ensure sterile conditions.
2. Thaw aliquots of biolaminin and add 120 µL of 5 µg/mL biolaminin to each glass cover slip. Leave the coated glass coverslips overnight at 4 °C.
3. Remove the excess of biolaminin and add 200 µL of hepatocyte differentiation medium as described below.

6. Preparation of hepatocyte differentiation media

1. Make 500 mL of hepatoblast knockout serum replacement (KSR)/dimethyl sulfoxide (DMSO) medium consisting of 76.4% knockout DMEM (KO-DMEM), 20% KSR, 0.5% L-glutamine, 1% non-essential amino acids (NEAA), 0.1% β-mercaptoethanol, 1% DMSO, and 1% penicillin-streptomycin (Pen/Strep).
2. Prepare 500 mL of hepatocyte maturation medium containing 1% L-glutamine, 10 µM hydrocortisone 21-hemisuccinate sodium salt (HCC), and 1% Pen/Strep.
3. Aliquot medium from the stock and add fresh hepatocyte growth factor (HGF) and oncostatin M (OSM) at final concentrations of 10 ng/mL and/or 20 ng/mL for each medium change.
   ​NOTE: Oncostatin M is a cytokine belonging to the interleukin 6 group of cytokines, important for hematopoiesis as well as for liver development.

7. Culturing hepatic cells differentiated from BD-PSCs

1. Put 3 x 10⁵ BD-PSCS cells into each well of a 4-well plate coated with biolaminin.
2. Place the 4-well plates in the incubator at 37 °C and 5% CO₂. Culture the cells for 5 days in KSR/DMSO hepatoblast medium that supports endodermal differentiation and change the medium every second day.
3. Switch to hepatocyte maturation medium on day 5 and culture the cells for an additional 7-10 days in the incubator at 37 °C and 5% CO₂. Change the medium every 48 h.

8. 3D spheroid hepatic differentiation

1. Count cells using a counting chamber.
2. Centrifuge BD-dedifferentiated cell suspension at 300 x g for 10 min at room temperature. Remove supernatant and resuspend BD-dedifferentiated cells in KSR/DMSO medium at 2 x 10⁶ cells/mL.
3. Pass the cells through a 40 µm cell strainer to ensure a single-cell suspension and to remove any additional debris.
4. Count cells using a counting chamber and prepare a sufficient volume for each cell seeding density in order to dispense the required volume per well. Prepare a gradient with top seeding of 1 x 10⁶ cells to a low seeding density of 4000 cells.
5. Dispense 100 µL of KSR/DMSO medium in 96 well-low attachment plates and add 100 µL of cell seeding dilution.
6. Place low attachment plate in the incubator at 37 °C and 5% CO₂ and culture them for 5 days.
7. Change 50% of the medium from days 3-4 after seeding when the spheroids are sufficiently compact.
8. On day 5, change the medium to hepatocyte maturation medium and culture the cells for an additional 7-10 days for further maturation. Change the medium every second day.

9. Immunofluorescence analysis of newly generated 2D liver cell cultures

1. Culture the cells according to the differentiation method described above for 4, 8, 15, and 24 days and remove media.
2. Incubate cells with pre-warmed fixative consisting of 4% paraformaldehyde in PBS for 10 min. Discard the fixative and wash the cells 2x with PBS for 5 min each. Immediately add 0.1% triton X-100 solution and permeabilize the cells for 5 min. Wash 2x with PBS.
3. Add blocking solution made of PBS and 5% BSA and place on rocker plate for 1 h at room temperature.
4. Dilute primary antibodies in dilution buffer 1% BSA/PBS as follows: albumin (ALB) 1:50, alpha-1 fetoprotein (AFP) 1:250, cytokeratin 18 (CK18) 1:50, hepatocyte nuclear factor 4 alpha (HNF4α) 1:1000 and transthyretin (TTR) 1:50. Use 50 µL of antibody dilution per well.
5. Incubate the cells for 1 h at room temperature. Then discard the antibody solution, wash the cells for 5 min, and repeat the wash step 3x.
6. Prepare the following secondary antibodies in dilution buffer: rabbit anti-chicken IgG (Texas red) 1:1000, goat anti-mouse IgG (488) 1:1000, and goat anti-rabbit (488) 1:500. Use 50 µL of antibody dilution per well and incubate the cells for 30 min at room temperature.
7. Wash the cells 3x with PBS for 5 min each and mount the coverslips with mounting media containing DAPI for microscopic analysis.

10. Live staining of newly formed liver spheroids

1. Carefully discard culture media while not touching the spheroids, add freshly made PBS with 0.1% triton X-100 solution, and incubate for 5 min to permeabilize the cells.
2. Wash the spheroids with the medium by slowly pipetting for 5 min, repeat 2x.
3. Incubate the spheroids with the primary antibodies ALB (1:50), AFP (1:250), CK18 (1:50), CYP2E1 (1:200), and CYP3A4 (1:200) prepared in PBS with 1% BSA for 1 h in 5% CO₂ incubator at 37 °C. Use 50 µL of antibody dilution per well.
4. Carefully remove the excess antibody solution and wash the spheroids with medium 3x.
5. Prepare the corresponding secondary antibodies goat anti-mouse IgG (Cy3), goat anti-mouse IgG (488), and rabbit anti-chicken IgG (Texas red) at a dilution of 1:1000 and 1:500 for goat anti-rabbit (488) in PBS in 1% BSA. Use 50 µL of antibody dilution per well and incubate for 20 min in the incubator at 37 °C and 5% CO₂.
6. Carefully wash 3x with medium and leave the plate for 30 min in the incubator at 37 °C and 5% CO₂ before performing fluorescence microscopy.

11. Examination of spheroids using a fluorescence microscope

1. Switch on the fluorescence light source 10 min before use, turn on the computer and open the imaging software.
2. Use an objective with 4x magnification, click on button 4x in the toolbar to have the correct scale bar selected, then place the 96-well plate on the stage center plate.
3. Turn the LED light source on, use the brightfield filter, and position the plate to the well of interest using the x-y-axis stage adjustment knob.
4. Change to camera light path, click the button Live in imaging software to visualize the image on the screen, and ensure the spheroid is centered using the x-y-axis knobs and focus using the coarse/fine focus knob.
   NOTE: The shape of the spheroids remains constant after applying live staining.
5. Choose the Brightfield Observation method in the toolbar, put exposure settings to automatic, and click the button Snapshot in the camera control panel to take a picture. Then save the picture as .vsi file using the appropriate name in the folder of interest.
6. Place ambient light shielding plate to turn off LED light, change to filter for B-excitation, choose 488 Observation method in the toolbar, open shutter, take a picture by clicking button Snapshot, close shutter, then save file as described above.
7. Repeat this with a filter for G-excitation using the appropriate observation method (Texas red or CY3). Then reiterate the whole procedure for each well of interest.
8. Return the plate to the 5% CO₂ incubator at 37 °C and culture it as described above.

결과

We successfully differentiated human BD-PSCs into endoderm/hepatic progenitor cells and hepatocytes by applying a two-step protocol. Morphological changes during the hepatic differentiation process are shown in Figure 1. BD-PSCs differentiate into hepatocytes going through three different stages. The first stage represents the differentiation into endodermal cells L4, the second, differentiation to hepatic progenitor cells (hepatoblast) L8, exhibiting a typical polygonal morphology, and the ...

토론

The liver is a major organ in the human body with many essential biological functions, such as the detoxification of metabolites. Due to severe liver failures like cirrhosis and/or viral hepatitis, there are nearly 2 million deaths per year worldwide. Liver transplantations rank second in solid organ transplantations worldwide, but only about 10% of the current need is met²².

Primary human hepatocytes (PHH) are often used to study liver toxicity. These cells can be main...

자료

Name	Company	Catalog Number	Comments
Albumin antibody	Sigma-Aldrich	SAB3500217	produced in chicken
Albumin Fraction V	Carl Roth GmbH+Co. KG	T8444.4
Alpha-1 Fetoprotein	Proteintech Germany GmbH	14550-1-AP	rabbit polyclonal IgG
Biolaminin 111 LN	BioLamina	LN111-02	human recombinant
CD45 MicroBeads	Miltenyi	130-045-801	nano-sized magnetic beads
Cell Strainer	pluriSelect	43-10040-40
CellSens	Olympus		imaging software
Centrifuge tubes 50 mL	Greiner Bio-One	210270
CEROplate 96 well	OLS OMNI Life Science	2800-109-96
CKX53	Olympus
Commercially available detergent	Procter & Gamble		nonionic detergent
CYP2E1-specific antibody	Proteintech Germany GmbH	19937-1-AP	rabbit polyclonal antibody IgG
CYP3A4	Proteintech Germany GmbH	67110-1-lg	mouse monoclonal antibody IgG1
Cytokeratin 18	DakoCytomation	M7010	mouse monoclonal antibody IgG1
DMSO	Sigma-Aldrich	D8418-50ML
DPBS	Thermo Fisher Scientific	14040091
FBS	Merck Millipore	S0115/1030B	Discontinued. Available under: TMS-013-B
Glass cover slips 14 mm	R. Langenbrinck	01-0014/1
GlutaMax 100x Gibco	Thermo Fisher Scientific	35050038	L-glutamine
Glutaraldehyde 25%	Sigma-Aldrich	G588.2-50ML
Goat anti-mouse IgG Cy3	Antibodies online	ABIN1673767	polyclonal
Goat anti-mouse IgG DyLight 488	Antibodies online	ABIN1889284	polyclonal
Goat anti-rabbit IgG Alexa Fluor 488	Life Technologies	A-11008
HCl	Sigma-Aldrich	30721-1LGL
HepatoZYME-SFM	Thermo Fisher Scientific	17705021	hepatocyte maturation medium
HGF	Thermo Fisher Scientific	PHG0324	human recombinant
HNF4α antibody	Sigma-Aldrich	ZRB1457-25UL	clone 4C19 ZooMAb Rbmono
Hydrocortisone 21-hemisuccinate (sodium salt)	Biomol	Cay18226-100
Knock out Serum Replacement - Multi Species Gibco	Fisher Scientific	A3181501	KSR
KnockOut DMEM/F-12	Thermo Fisher Scientific	12660012	Discontinued. Available under Catalog No. 10-828-010
MACS Buffer	Miltenyi	130-091-221
MACS MultiStand	Miltenyi	130-042-303	magnetic stand
MEM NEAA 100x Gibco	Thermo Fisher Scientific	11140035
Mercaptoethanol	Thermo Fisher Scientific	31350010	50mM
MiniMACS columns	Miltenyi	130-042-201
Nunclon Multidishes	Sigma-Aldrich	D6789	4 well plates
Oncostatin M	Thermo Fisher Scientific	PHC5015	human recombinant
Paraformaldehyde	Sigma-Aldrich	158127
PBS sterile	Carl Roth GmbH+Co. KG	9143.2
Penicillin/Streptomycin	Biochrom GmbH	A2213	10000 U/ml
PS 15ml tubes sterile	Greiner Bio-One	188171
Rabbit anti-chicken IgG Texas red	Antibodies online	ABIN637943
Roti Cell Iscoves MDM	Carl Roth GmbH+Co. KG	9033.1
Roti Mount FluorCare DAPI	Carl Roth GmbH+Co. KG	HP20.1
Roti Sep 1077 human	Carl Roth GmbH+Co. KG	0642.2
Transthyretin antibody	Sigma-Aldrich	SAB3500378	produced in chicken
Triton X-100	Thermo Fisher Scientific	HFH10	1%