Construction, Characterization, and Regenerative Application of Self-Assembled Human Mesenchymal Stem Cell Aggregates

Summary

This study describes a method to construct aggregates based on the self-assembly of human mesenchymal stem cells and identifies the morphological and histological characteristics for the regenerative treatment of cranial bone defects.

Abstract

Mesenchymal stem cells (MSCs), characterized by their self-renewal ability and multilineage differentiation potential, can be derived from various sources and are emerging as promising candidates for regenerative medicine, especially for regeneration of the tooth, bone, cartilage, and skin. The self-assembled approach of MSC aggregation, which notably constructs cell clusters mimicking the developing mesenchymal condensation, allows high-density stem cell delivery along with preserved cell-cell interactions and extracellular matrix (ECM) as the microenvironment niche. This method has been shown to enable efficient cell engraftment and survival, thus promoting the optimized application of exogenous MSCs in tissue engineering and safeguarding clinical organ regeneration. This paper provides a detailed protocol for the construction and characterization of self-assembled aggregates based on umbilical cord mesenchymal stem cells (UCMSCs), as well as an example of the cranial bone regenerative application. The implementation of this procedure will help guide the establishment of an efficient MSC transplantation strategy for tissue engineering and regenerative medicine.

Introduction

Mesenchymal stem cell (MSC) condensation is an essential stage to ensure the normal growth and development of the body in early organogenesis^1,2, especially in the formation of bone, cartilage, teeth, and skin^1,3,4. In the last few decades, tissue engineering therapies using cultured postnatal MSCs combined with biodegradable scaffolds have made important advances in osteogenic⁵ and cartilaginous regeneration⁶. However, the use of scaffolds may have some disadvantages, such as immune rejection, as well as low cellular affinity and plasticity⁷. In this regard, we have investigated the feasibility of applying a spheroid cell culture method to provide scaffold-free self-assembled aggregates mimicking the developing condensation phenomenon, which contain only MSCs and the deposited extracellular matrix (ECM)⁸. The formation of aggregates increases applicative plasticity to match the defect shape and avoids scaffold implantation and digestion by proteolytic enzymes to harvest MSCs for transplantation⁹.

MSC aggregates have been used widely for regeneration of the bone, dental pulp, periodontium, and skin¹⁰, among other tissue and organs. Many different types of MSCs can be selected as candidates for seed cells, including but not limited to bone marrow MSCs (BMMSCs), umbilical cord MSCs (UCMSCs), adipose tissue-derived stromal cells (ADSCs), and dental MSCs (e.g., dental pulp mesenchymal stem cell [DPSCs], mesenchymal stem cells from the deciduous teeth [SHED]¹¹, and periodontal mesenchymal stem cells [PDLSCs])¹². Many technologies for three-dimensional cell clusters have been developed in the past decade, including assisted and self-assembled aggregation. However, assisted aggregation approaches are often weak in producing ECMs and forming homogeneous and tight aggregates, and are therefore not suitable for mimicking physiological conditions^13,14,15. Moreover, some assisted aggregation methods require cell-material interactions to form stable structures^16,17,18,¹⁹, whereas this self-assembled aggregation method is generally available for a wide range of MSCs. Notably, in our recent clinical trials, MSC aggregates have been successfully used to regenerate the pulp-dentin complex and the periodontium after implantation into injured human incisor teeth, which have achieved de novo tissue regeneration with physiological structure and function^20,21.

This paper provides a thorough procedure for MSC aggregate construction and characterization, as well as in vivo transplantation. This approach will attract the attention of researchers when they aim to repair defects in tissues, such as the teeth, bone, cartilage, and skin, based on stem cell applications. This method is simple, convenient, and completely composed of cells and ECM without additional scaffolds, which can be cultured for a long time to obtain dense and stable aggregates²². Meanwhile, the aggregates cultured in this way are rich in ECM, which mimics the developing niche for these high-density cells and thus promotes tissue regeneration²³. The construction process can be divided into two stages: cell preparation and culture, and self-assembled formation and harvest of cell aggregates. The characterization of aggregates includes morphological identification via an inverted optical microscope and a scanning electron microscope (SEM), and histological analysis via hematoxylin and eosin (HE) and Masson's staining. The formed aggregates were demonstrated for regenerative implantation to repair the cranial bone defect. The implementation of this procedure will help guide the establishment of an efficient MSC transplantation strategy for tissue engineering and regenerative medicine.

Protocol

NOTE: All animal procedures were approved by the Animal Care and Use Committee of the Fourth Military Medical University and performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Cryopreserved human UCMSCs that were obtained from a commercial source were used for the present study (see Table of Materials). The use of human cells was approved by the Ethics Committee of the Fourth Military Medical University. UCMSCs were taken as an example to describe the procedure. The cranial defect was taken as an example for showing the need of repair to describe the implantation procedure. All the experiments were repeated three times.

1. Construction of UCMSC aggregates

1. Preparation and culture of UCMSCs
   1. Add 10 mL of prewarmed phosphate-buffered saline (PBS) into a new 15 mL centrifugal tube in a sterile biosafety cabinet.
   2. Remove the freezing tube containing frozen UCMSCs from liquid nitrogen and quickly place it into a 37 °C water bath. Shake the tube gently to facilitate rapid and thorough cell thawing.
      CAUTION: The whole procedure must be finished within 1 min, because dimethyl sulfoxide (DMSO) in the freezing solution may cause cytotoxicity.
   3. Slightly suspend the cells in the freezing tube and transfer all the contents into the centrifuge tube containing 10 mL of PBS (step 1.1.1). Centrifuge the sample at 100 × g for 5 min. Discard the supernatant and wash the cells with 10 mL of PBS.
   4. Centrifuge the sample at 100 × g for 5 min. Discard the supernatant, and gently resuspend the cells with complete alpha-minimum essential medium (α-MEM) containing 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, and 584 mg/L glutamine.
   5. Seed the resuspended cells in 10 cm culture dishes (often one to two dishes for one tube, depending on the total amount of cells after centrifugation, at a density of 5 × 10⁶ cells per dish). Add complete α-MEM medium in the dishes to a total volume of 10 mL.
   6. Gently shake the dishes to distribute the cells. Incubate the dishes at 37 °C with 5% CO₂ in a humidified chamber. After overnight incubation,discard the supernatant medium and add 5 mL of PBS 1-2x to remove the nonadherent cells. Add 10 mL of fresh complete α-MEM medium. Change the culture media every 2 days.
   7. For cell passaging, when the cells reach 80%-90% confluence, discard the supernatant and wash the cells with 5 mL of PBS to remove the medium thoroughly. Incubate the cells in 2 mL of 0.25% trypsin-1 mM ethylenediaminetetraacetic acid (EDTA) at 37 °C for 2 min.
   8. When the cells become round with broken intercellular junctions under a microscope, separate the cells from the dishes without too much disruption of the cell structure. Neutralize the trypsin by adding 4 mL of complete α-MEM medium.
   9. Collect the cells from the dishes repeatedly and gently by pipetting in a fresh 15 mL centrifuge tube. Centrifuge the sample at 100 × g for 5 min. Discard the supernatant, resuspend the cells with complete α-MEM medium, and seed them in fresh culture dishes or well plates. Change the culture media every 2 days.
      NOTE: According to the size of the defect area, a 25 mm³ defect requires the use of a well of 6-well plate cells cultured to form aggregates.
2. Formation and harvest of aggregates
   1. Prepare cell aggregate-inducing media by adding 50 µg/mL vitamin C into α-MEM with 10% FBS, 1% penicillin-streptomycin, and 584 mg/L glutamine.
      CAUTION: Direct light exposure should be avoided when using vitamin C, as vitamin C oxidizes when exposed to light and becomes ineffective.
   2. When the cells in step 1.1.12 reach 95% confluence (Supplemental Figure S1), change the media to cell aggregate-inducing media; change fresh cell aggregate-inducing media every 2 days.
   3. After 7-10 days of inducing (the length of inducing time depends on the stem cell types and is often ~7 days for the edge of UCMSCs), look for shaped aggregates under the microscope, with thick edges separated from the dish bottom.
   4. Discard the culture media and gently wash the cells 2x with PBS. Gently push the aggregates from the edge to the middle using small sterile forceps, folding the aggregates.
   5. On some occasion, UCMSCs can spontaneously curl toward the center to form a solid condensation. This structure is equivalent to the aggregate obtained in step 1.2.4.

2. Morphological identification of aggregates

1. General and microscopic observation
   1. Observe that the aggregates are dense and integrated and are not easily damaged by mechanical forces, such as pulling during detachment and transplantation.
   2. Under a microscope, look for a membrane-like structure of a certain thickness with woven pattern.
2. Live/dead cell staining
   1. Seed the cells in 6-well plate at a density of 1 × 10⁶ cells per well.
   2. Prepare 2 µM calcein AM and 4 µM EthD-1 working solution and using PBS.
   3. Add 0.3% triton to the cells for 5 min, wash with PBS three times to induce the cells to death, as a control for staining.
   4. Wash the cells, aggregates, and dead-inducing cells with PBS twice.
   5. Add 100 µL of PBS and 100 µL of prepared staining solution in step 2.2.2 to each well.
   6. Incubate the sample for 30 min at RT. Wash the sample with PBS twice.
   7. Add 200 µL of 10 µg/mL Hoechst to each well and incubate the sample for 5 min at RT. Wah the sample with PBS twice.
   8. Observe under a fluorescence microscope.
3. SEM observation
   1. Seed and induce cell the aggregates obtained in step 1.2.4 or 1.2.5. Wash the cells with PBS after the aggregates are formed in step 1.2.4. or 1.2.5.
   2. Fix the cells using glutaraldehyde for at least 4 h.
   3. Wash the samples with PBS, then dehydrate through a gradient concentration of ethanol from 30%, 50%, 70%, 80%, 90%, to 100%, each for 5 min. Treat 2x with anhydrous ethanol.
   4. Immerse the aggregates in 3 mL of hexamethyldisilazane for 30 min. Aspirate all fluid and naturally dry the samples thoroughly.
      CAUTION: The whole process needs to be carried out in a fume hood because hexamethyldisilazane is volatile and highly toxic.
   5. Stick the dried samples onto the sample table with double-sided carbon tape and coat the surface with metal. Store the sample at room temperature (RT) for several days.
   6. Observe under an SEM.
      1. Replace the sample inside the machine. Adjust the machine voltage to 5 k, and the focal length to ~12 mm. Adjust the focus bar until a clear picture appears.
      2. Capture the images at low magnification. Adjust the magnification and refocus to obtain a higher magnification image.
         ​CAUTION: The surface of the aggregates cannot be touched before observation to avoid destruction of surface morphology.

3. Histological analysis of aggregates

1. Fixation and sectioning of samples
   1. Fix the aggregates obtained in step 1.2.4 or 1.2.5with 4% paraformaldehyde at 4 °C for at least 24 h. After the fixation, wash with PBS and place the sample into embedding cassettes overnight to rinse with running water.
   2. Dehydrate the samples using automatic dehydration machines through gradient ethanol, xylene, and paraffin, following the manufacturer's instructions. The whole process is as follows: 85% ethanol for 2 h at RT, 95% ethanol for 2 h at RT, 95% ethanol for 2 h at RT, 100% ethanol for 2 h at RT, 100% ethanol for 1.5 h at RT, xylene for 45 min at RT, xylene for 45 min at RT, paraffin for 30 min at RT, paraffin for 1.5 h at RT, and finally paraffin for 2 h at 60 °C.
   3. Embed the samples with paraffin. Prepare approximately 5 µm thick sections, adhere them to cationic slides, and dry the slides for 2 h at 45 °C.
   4. Dewax sequentially through xylene for 15 min, twice, and 100%, 90%, 80%, and 70% ethanol (each for 5 min).
   5. Slowly rinse the slides 3x with running water for 5 min.
2. HE staining
   1. Stain with hematoxylin for 3-5 min, rinse with water, and carefully remove excess fluid around the tissue.
   2. Fractionate with ethanol containing 1% hydrochloric acid for 3-5 s and rinse with running water, during which the color becomes a little lighter and blue. Remove excess water around the tissue.
   3. Stain with eosin for 1 min, rinse with running water, and remove excess fluid around the tissue.
   4. Dehydrate with 70%, 80%, 90%, and 100% ethanol for 10 s each, xylene for 15 min, twice, and dry naturally in the fume hood before sealing the sheet.
   5. Add neutral resin drops and seal the slides with coverslips. Observe under a light microscope.
3. Masson's trichrome staining
   1. Stain with Weigert's iron hematoxylin for 5 min, rinse with water, and wipe dry.
   2. Fractionate with ethanol containing 1% hydrochloric acid for 3-5 s and rinse with running water, during which the color becomes a little lighter and blue. Wipe the surface dry.
   3. Stain with Lixin red acidic magenta solution for 5-10 min and rinse quickly with distilled water.
   4. Treat with phosphomolybdic acid aqueous solution for 3-5 min.
   5. Directly re-stain with aniline blue solution for 5 min without washing with water.
   6. Treat with 1% glacial acetic acid for 1 min.
   7. Dehydrate with 70%, 80%, 90%, and 100% ethanol for 10 s each, xylene for 15 min, twice, and dry naturally in the fume hood before sealing the sheet.
   8. Add neutral resin drops and seal the slides with coverslips. Observe under a light microscope.

4. Implantation

1. Anesthetize the nude mouse by an intraperitoneal injection of 50 mg/kg pentobarbital sodium.
   ​NOTE: Confirm the mice are properly anesthetized based on the absence of corneal reflex and limb reaction when the footpad is pinched. Apply eye ointment to prevent dryness.
2. Cut out a transverse incision (~3-4 cm) in the skin behind the ears of the nude mouse with sterile small scissors after disinfection with iodophor.
3. Pull the skin forward to expose the cranium. Lightly draw the edges of the planned defect area with an 11# blade and remove the bone fragment. The defect area is a circular area of approximately 4 mm in diameter. Use saline to flush the wound and provide adequate hemostasis.
4. Dip the aggregates obtained in step 1.2.5 or 1.2.6 with sterile gauze to absorb surface moisture before implantation. Place the aggregates into the area and slightly pressurize to fill the defect.
5. Replace the skin after pulling to make sure the implant is fixed. Close the incision with 1/2, 4 × 10 angled stitches and 4-0 sutures.
6. After the surgery, place the animals on the rewarming blanket until they are active and then return them into the cage.
   NOTE: The animals must not be left unattended until they regain sufficient consciousness to maintain sternal recumbency. The animals that have undergone surgery are not returned to the company of other animals until fully recovered.

Results

Aggregates can be successfully constructed from UCMSCs according to the experimental workflow (Figure 1). The quality of aggregates must be evaluated prior to use, via morphological observation and histological analysis. The lamellar structure formed should be complete and dense, with the cells interlaced to form a woven pattern by microscopic observation (Figure 2A). Edge curling can be discovered during aggregation; overcurling edges indicate unsucces...

Discussion

With the advances of tissue engineering biotechnology, strategies to construct an implantable structure with high plasticity and containing long-term-surviving cells that can achieve optimal regeneration have been the focus of many scientists. There are a variety of current implantation methods of MSCs, such as cell-only methods, scaffolds complemented with cytokines^6,24, or the combination of stem cells and scaffolds⁵. This paper presents...

Materials

Name	Company	Catalog Number	Comments
0.25% Trypsin-EDTA (1x)	Sigma	T4049	Cell passage
Automatic Dehydration Machine	LEICA	ASP200s	Dehydrate aggregate
Centrifuge	Eppendorf	5418R	Centrifugation
Centrifuge tube	Thermo Nunc	339650	Centrifugation
Culture dish	Thermo	150466	Culture of UCMSCs
Ethanol	SCR	10009218	Dehydrate aggregate
Fatal bovine serum	Sijiqing	11011-8611	Culture of UCMSCs
Forcep	JZ	JD1080	Harvest aggregate
Glutaraldehyde	Proandy	10217-1	Fixation of aggregate
Hematoxylin and Eosin Staining Kit	beyotime	C0105S	HE staining
Hexamethyldisilazane	SCR	80068416	Dry aggregate surface
Hoechst33342	Sigma	14533	Cell nuclei stain
L-glutamine	Sigma	G5792	Culture of UCMSCs
Live/dead Viability/Cytotoxicity Kit	Invitrogen	L3224	Live/dead cell stain
Masson's Staining Kit	ZHC	CD069	Masson Staining
Minimum Essential Medium Alpha basic (1x)	Gibco	C12571500BT	Culture of UCMSCs
Paraffin	Leica	39601006	Tissue embedding
Paraformaldehyde	Saint-Bio	D16013	Fixation of aggregate
PBS (1x)	Meilunbio	MA0015	Resuspend and purify UCMSCs
Penicillin/Streptomycin	Procell Life Science	PB180120	Culture of UCMSCs
Pentobarbital sodium	Sigma	P3761	Animal anesthesia
Polysporin	Pfizer		Prevent eye dry
Scanning Electron Microscope	Hitachi	s-4800	SEM observation
Scissor	JZ	Y00030	Animal surgical incision
Six-well plate	Thermo	140675	Culture of UCMSCs
Stitch	Jinhuan	F603	Close wounds
Suture	Xy	4-0	Close wounds
Thermostatic equipment	Grant	v-0001-0005	Water bath
UCMSCs	Bai'ao	UKK220201	Commercially UCMSCs
Vitamin C	Diyibio	DY40138-25g	Aggregate inducing
Xylene	SCR	10023418	Dehydrate aggregate